Elucidation of the Mechanism by Which Protein Kinase Ck2 Promotes Cell Survival

Home , Stuart Duncan

ELUCIDATION OF THE MECHANISM BY WHICH PROTEIN KINASE CK2 PROMOTES CELL SURVIVAL

(Spine title: Investigating the role of CK2 in cell survival)

(Thesis format: Integrated-Article)

James S. Duncan

Graduate Program in Biochemistry

A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

School of Graduate & Postdoctoral Studies The University of Western Ontario London, Ontario, Canada

395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada

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CERTIFICATE OF EXAMINATION

Supervisor Examiners

Dr. David Litchfield Dr. Caroline Schild-Poulter

Supervisory Committee Dr. Joseph Torchia

Dr. David Haniford Dr. James Koropatnick

Dr. David Edgell Dr. Lynn Megeney

The thesis by

James Stuart Duncan

entitled:

Elucidation of the Mechanism By Which Protein Kinase CK2 Promotes Cell Survival

is accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Date Chair of the Thesis Examination Board

ii ABSTRACT

Elevated CK2 activity has been associated with the malignant transformation of several tissues, and is associated with aggressive tumor behavior. While the precise roles of CK2 in tumorigenesis remain incompletely understood, mounting evidence suggests a role for CK2 in the protection of cells from apoptosis via the regulation of tumor suppressor and oncogene activity. Consequently, CK2 has emerged as a potential therapeutic target, and strategies to inhibit CK2 have been ongoing in pre-clinical trials.

In the present studies, an unbiased evaluation of CK2 inhibitors TBB, TBBz and DMAT was carried out to elucidate the mechanism of action, as well as inhibitor specificity of these compounds. Utilizing a chemo-proteomic approach in conjunction with inhibitor-resistant mutant studies, isoforms CK2a and CK2a' were identified as bona fide targets of TBB, TBBz and DMAT in cells. However, a number of putative off- target inhibitor interactions were identified, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme Quinone Reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors, as well as drug specificity, which will assist in the development of more specific next-generation CK2 inhibitors.

The convergence of protein kinases and caspase signaling pathways has become increasingly evident, as phosphorylation of a number of caspase substrates within the caspase recognition motif has been shown to prevent caspase cleavage. To investigate the global role of phosphorylation in the regulation of caspase signaling, a novel sequence- based protein identification targeting strategy, (sPITS) was employed. Intriguingly, the constitutively active and oncogenic protein kinase CK2 shares an overlapping requirement with caspases for an acidic consensus sequence, therefore, a comprehensive identification of overlapping CK2/caspase targets was carried out. A number of proteins involved in cell survival were identified, including pro-caspase-3, which was shown to be phosphorylated by CK2 preventing its caspase-dependent activation in cells. Validation of the phosphorylation-dependent protection of pro-caspase-3, as well as identification of numerous candidate CK2/caspase targets in the present studies, support a role for phosphorylation as a global mechanism of regulation of caspase signaling pathways.

iii Keywords: Protein kinase CK2, tumorigenesis, phosphorylation, kinase inhibitors, chemo-proteomics, bioinformatics, apoptosis, caspases, mass spectrometry, 2D gel electrophoresis

iv CO-AUTHORSHIP STATEMENT

The following thesis contains material from previously published manuscripts. David Litchfield is a co-author on all presented papers and was responsible for supervising James S. Duncan during his thesis. For all chapters in this thesis in which a version has been published, James S. Duncan wrote the first draft of the manuscript and David Litchfield played a major role in the editing and revisions of the manuscripts.

James Duncan carried out all of the chemo-proteomics experiments described in Chapter 2, prepared all of the figures and wrote the manuscript for publications related to the work of Chapter 2. Laszlo Gyenis and John Lenehan contributed in the preparation and running of a portion of the numerous 2D gels utilized in Chapter 2. Tim Haystead was instrumental in supplying the ATP-sepharose required for the competition assays used in the chemo-proteomics approach, while Lee Graves contributed valuable insight into the proteomic aspects of Chapter 2. Maria Bretner provided a variety of CK2 inhibitors utilized in the chemo-proteomic experiments.

James Duncan carried out all of the experiments related to the investigation of phosphorylation as a global mechanism of regulation of caspase signaling, prepared all of the figures and wrote the manuscript for publications related to the work of Chapter 3 and Chapter 4. Greg Gloor was essential in writing and carry out the peptide match program identifying the overlapping CK2/caspase targets from the human proteome, while Shawn Li and Chenggang Wu provided the peptide arrays and fluorescein/biotin labeled peptides. Validation of CSI using mass spectrometry was carried out by James Duncan and Kelly Duncan. Jake Turowec helped purify caspases required for in vitro studies and CSI, as well as performed caspase cleavage assays.

v DEDICATION

/ dedicate this thesis to my loving wife Dr. Kelly Duncan. I would not have made it this far without you. ACKNOWLEDGMENTS

First and foremost, I would like to extend my sincerest appreciation to my supervisor, David Litchfield, for all of his guidance and support over the last five years. I also wanted to say thank you for giving me the opportunity to travel to Europe for a conference last summer, it was a very memorable experience.

I would like to thank my family: my mom and dad; my sister, Angie and her husband Jeff; my brother-in-law, Brett; and my father- and mother-in-law, Mike and Pat. Their love and support mean the world to me.

Sincere thanks to my friends John Lenehan (aka. ICEMAN) and Ryan Mohan who were always their to discuss current topics over some fine beers at the grad club. I'd also like to acknowledge all of my friends in the Litchfield lab over the last five years and the Biochemistry Department, for all of the good times over the years. The biochemistry functions wouldn't have been as fun without you!

Many thanks to the following people. My research project would not have been possible without you.

• Dr. Greg Gloor for all your helpful discussions regarding the CK2/caspase project

• Dr. Shawn Li for critical input into the design and execution of sPITS

• Dr. Tim Haystead and Dr. Lee Graves for guidance on experimental design regarding the chemo-proteomics project

• Jacob Turowec for all the help with preparing and carry out caspase cleavage assays

• Chenggang Wu for preparation of the peptide arrays and fluorescein and biotin labeled peptides

• Kystina Jurcic for helping with the protein identification regarding the CK2 inhibitors project

vii • Victoria Clarke for all of her help with preparing DNA preps throughout the five years

• Chris Ward for contributions in preparing the numerous 2D gels used in the chemo-proteomics studies

• CIHR-strategic training program, CIHR Canadian Graduate Scholarship, Ontario Graduate Scholarship, Ontario Graduate Scholarship Science Technology, the Department of Biochemistry and the Faculty of Science for various funding opportunities

A very special thanks goes to my wife Kelly for always being there when I needed you!

viii TABLE OF CONTENTS

CERTIFICATE OF EXAMINATION ii

ABSTRACT iii

CO-AUTHORSHIP STATEMENT v

DEDICATION vi

ACKNOWLEDGMENTS vii

TABLE OF CONTENTS ix

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF APPENDICES xx

LIST OF ABBREVIATIONS AND DEFINITIONS xxi

CHAPTER 1 INTRODUCTION 1

1.1 The druggable kinome 1

1.2 Protein kinase CK2 1

1.3 CK2 promotes tumorigenesis 3

1.3.1 CK2 regulates the activity and stability of tumor suppressor proteins 5

1.3.2 CK2 promotes cell survival through the regulation of oncogenes 6

1.4 CK2-dependent pathways involved in development of cancer 7

1.4.1 NF-kB pathway 7

ix 1.4.2 Wnt signaling pathway 8

1.4.3 PI3'K pathway 11

1.5 An emerging anti-apoptotic role for CK2 11

1.5.1 Evidence of sensitization of cells to apoptosis following CK2 inhibition 13

1.5.2 Regulation of apoptotic machinery by CK2 14

1.6 Exploitation of strategies to inhibit CK2 function 15

1.7 Knowledge-based design of CK2 inhibitors 17

1.8 Characterization of CK2 inhibitors in vivo 18

1.9 Identification of CK2 targets utilizing chemical inhibitors 20

1.10 Validation of CK2 targets "identification of bona fide targets" 22

1.11 Scope of Thesis 25

1.12 References 29

CHAPTER 2 AN UNBIASED EVALUATION OF CK2 INHIBITORS BY CHEMO-PROTEOMICS: CHARACTERIZATION OF INHIBITOR EFFECTS ON CK2 AND IDENTIFICATION OF NOVEL INHIBITOR TARGETS 43

2.1 Introduction 43

2.2 Materials and Methods 46

2.2.1 Cell culture and transfections 46

2.2.2 Cloning of CK2ccR and CK2cc'R 47

2.2.3 Flow cytometry 47

x 2.2.4 Western analysis 47

2.2.5 Immunoprecipitations 48

2.2.6 ATP-Sepharose affinity chromatography 48

2.2.7 2D gel electrophoresis 49

2.2.8 2D gel electrophoresis quantification and analysis 50

2.2.9 Sample preparation and mass spectrometry 50

2.3 Results 51

2.3.1 Effect of CK2 inhibitors on cell viability: Evidence for unique modes of drug action 51

2.3.2 Employing inhibitor-resistant CK2 mutants to test drug specificity 54

2.3.3 Identification of novel inhibitor interactors: Evidence for off-target effects 59

2.4 Discussion 63

2.5 References 68

CHAPTER 3 DECIPHERING CELLULAR DESCISIONS OF LIFE AND DEATH: CONVERGENCE OF PROTEIN KINASES AND CASPASE SIGNALING 73

3.1 Introduction 73

3.2 Materials and Methods 76

3.2.1 Identification of overlapping CK2 and caspase targets using bioinformatics 76

3.2.2 Phosphorylation of peptide arrays by CK2 76

XI 3.2.3 Caspase Substrate Identification (GST) 77

3.2.4 Mass spectrometry analysis of peptide cleavage 78

3.2.5 Caspase-3 activity assay 78

3 Results 78

3.3.1 Functional characterization of overlapping CK2/caspase targets 79

3.3.2 Phosphorylation of putative targets on peptide arrays by CK2 80

3.3.3 Identification of caspase targets using Caspase Substrate Identification (CSJ) 83

3.3.4 Validation of caspase-mediated cleavage using mass spectrometry in conjunction with CSI 83

3.3.5 Global identification of candidate CK2/caspase targets sensitive to caspase cleavage 87

3.3.6 Phosphorylation of the P2 and PI' residue of caspase-3 peptides prevents caspase cleavage 91

4 Discussion 91

3.4.1 Development and exploitation of throughput strategies to study complex signaling networks 93

3.4.2 Identification of candidate CK2 targets using peptide array target screens 94

3.4.3 Identification of CHD8 as a promising overlapping CK2/caspase target using sPITS 95

3.4.4 Phosphorylation of the P2 or PI' within proteolytic activation cleavage site of caspase-3 prevents cleavage 98

5 References 101

xn CHAPTER 4 REGULATION OF PRO-CASPASE-3 PROTEOLYTIC ACTIVATION BY CK2: A MECHANISM FOR PROTECTION OF CELLS FROM APOPTOSIS 106

4.1 Introduction 106

4.2 Materials and Methods 109

4.2.1 Cell culture and transfections 109

4.2.2 Generation of pro-caspase-3 mutants and purification 109

4.2.3 Knockdown and inhibition studies 109

4.2.4 Immunoblot analysis 110

4.2.5 Immunoprecipitations 110

4.2.6 Phosphorylation of caspase-3 by CK2 in vitro Ill

4.2.7 32P cell labeling and TBB treatment Ill

4.2.8 Cleavage of pro-caspase-3 by caspase-8 and -9 112

4.2.9 Assay of caspase-3 activity in cell lysates 113

4.3 Results 113

4.3.1 Interference with CK2 activity results in spontaneous apoptosis 113

4.3.2 Phosphorylation of pro-caspase-3 by CK2 protects from cleavage by caspase-8 and -9 115

4.3.3 Phosphorylation of Thrl74 and Serl76 by CK2 protect pro-caspase-3 from caspase-mediated cleavage 118

4.3.4 Interaction of CK2 and pro-caspase-3 in cells; evidence for CK2- dependent phosphorylation of pro-caspase-3 120

xiii 4.4.4 Interference with CK2 activity results in an increase in activation of caspase-3 during apoptosis 122

4.4 Discussion 124

4.5 References 129

CHAPTER 5 CONCLUSIONS AND PERSPECTIVES: EVOLVING METHODS TO ELUCIDATE THE MOLECULAR MECHANISMS OF CK2 IN TUMORIGENESIS 134

5.1 The importance of global protein target identification strategies to investigate the role of CK2 in the development of cancer 134

5.2 Identification of CK2 inhibitor off-targets using chemo-proteomics: A re- evaluation of the use CK2 inhibitors to study CK2 function 136

5.3 Generation of analog-sensitive CK2 gatekeeper mutants to identify global CK2 targets 140

5.4 Convergence of protein kinases and caspase signaling pathways: Evidence for a global role of phosphorylation in the regulation of caspase signaling 145

5.5 Regulation of caspase-3 proteolytic activation by CK2: a mechanism for protection of cells from apoptosis 146

5.6 Perspectives: A working model for CK2 in the negative regulation of caspase signaling 149

5.7 Conclusion 151

5.8 References 155

APPENDIX A 160

xiv APPENDIX B 185

CURRICULUM VITAE 193

xv LIST OF TABLES

Table Description Page

1-1 A comparison of the efficacy of CK2 inhibitors 18

2-1 Identification of putative off-target CK2 inhibitor interactions 61

3-1 Proteins identified in peptide match search with an overlapping CK2 and caspase recognition motif cleaved by caspases in cells 79

3-2 Proteins identified from peptide match screen with an overlapping CK2 and caspase recognition motif previously reported to be phosphorylated in cells 82

3-3 Identification of candidate CK2/caspase peptide sequences sensitive to caspase-mediated cleavage 88

3-4 Members of the Chromodomain Helicase DNA binding protein (CHD) family phosphorylated at a CK2 consensus sites in cells 99

4-1 Proteins protected from caspase-mediated cleavage by CK2 phosphorylation 108

A-l Proteins identified using a peptide match program that contained an overlapping CK2 consensus for phosphorylation and a caspase cleavage recognition motif. 160

A-2 Peptides containing an overlapping CK2/caspase recognition sequence phosphorylated by GST-CK2oc on peptide arrays 177

B-l Putative CK2 inhibitor-biomarkers identified using functional proteomics 189

xvi LIST OF FIGURES

Figure Description Page

1-1 The molecular role of CK2 in the progression of tumorigenesis 4

1-2 The role of CK2 in the perpetuation of the PI3'K signaling pathway 12

2-1 Chemical structure of ATP and commercially-available CK2 inhibitors 45

2-2 CK2 inhibitor-dependent induction of caspase-mediated apoptosis 52

2-3 Concentration- and duration-dependent induction of apoptosis following CK2 inhibitor treatment 53

2-4 Employing inhibitor-resistant CK2a mutants to test drug specificity 55

2-5 Employing inhibitor-resistant CK2cc' mutants to test drug specificity 56

2-6 Apoptosis associated with TBBz and DMAT treatment was independent of CK2 activity 58

2-7 Exploiting an unbiased chemo-proteomic approach to identify CK2 inhibitor interactions 60

2-8 Concentration-dependent inhibitor interaction with CK2 62

2-9 Identification of a novel TBBz and DMAT off-target, Quinone Reductase, (QR2) 64

3-1 Schematic of sPITS: a novel strategy used to identify global overlapping protein kinase and protease targets 75

3-2 Functional characterization of proteins identified with an overlapping CK2/caspase consensus sequence 81

3-3 Caspase Substrate Identification (CSI), a novel method to detect caspase cleavage 84

xvn 4 Validation of CSI to detect caspase cleavage 86

5 Detection of caspase cleavage of known cellular caspase targets using CSI 89

6 Identification of candidate CK2/caspase-3 targets using CSI 90

7 Addition of a phosphate group at the P2 or PI' residue within the proteolytic activation site of pro-caspase-3 peptides prevents caspase- mediated cleavage 92

8 Alignment of predicted overlapping CK2 phosphorylation site and caspase cleavage motif in HDAC4, Aven, CHD4 and CHD8 96

1 Spontaneous apoptosis is associated with interference with CK2 function 114

2 Phosphorylation of pro-caspase-3 by CK2 results in protection from caspase-mediated cleavage 116

3 Phosphorylation of pro-caspase-3 at Thrl74 and Serl76 protects from caspase-mediated cleavage 119

4 CK2 interacts with and phosphorylates pro-caspase-3 in cells 121

5 Inhibition of CK2 facilitates the maturation of active caspase-3 123

6 A model for the regulation of caspase-3 signaling by CK2 125

1 Utilizing a functional proteomic approach to identify CK2 substrates in cells 138

2 Alignment of gatekeeper residue in CK2a/a' with CDK family members 142

3 The use of a chemical genetic approach in conjunction with functional proteomics to identify CK2 substrates 144

xviii The use of SILAC to identify the dynamic relationship between CK2 and caspases during apoptosis 150

The global role of phosphorylation in the regulation of caspase signaling 153

Identification of CK2 inhibitor-dependent changes in phosphorylation using functional proteomics 188

CK2 gatekeeper mutants express and retain kinase activity in cells 190

Mutation of the CK2 phosphorylation sites in pro-caspase-3 generates a non-cleavable form of pro-caspase-3 191

Cleavage of CK2a by caspase-3 in vitro 192

xix LIST OF APPENDICES

Appendix A 160

Appendix B 185

xx LIST OF ABBREVIATIONS AND NOMENCLATURE

APC Adenomatous polyposis coli AS protein kinase Analog sensitive protein kinase ATP Adenosine triphosphate bp Base pair BSA Bovine serum albumin CDK7 Cyclin-dependent kinase 7 CHD Chromodomain Helicase DNA binding protein CK2 Protein kinase CK2 or casein kinase 2/II CK2a Catalytic isoform of CK2 CK2a' Catalytic isoform of CK2 CK2p Regulatory subunit of CK2 CK2/caspase Candidate target with an overlapping CK2 and caspase recognition motif CK2-GM Gatekeeper mutants of CK2 CSI Caspase substrate identification DMAT 2-Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole; DOC Deoxycholate DRB 5,6-Dichlorobenzimidazoleribofuranoside DYRK Dual specificity tyrosine phosphorylation-regulated kinase EEF1D Eukaryotic elongation factor-1 delta FITC Fluorescein isothiocyanate FL N-terminal fluorescein labeled peptide FL/Biotin peptide N-terminal labeled fluorescein and C-terminally biotin labeled peptide GAM Goat anti-mouse GAR Goat anti-rabbit GFP Green fluorescent protein GST Glutatione-S-transferase HA YPYDVPDY epitope

xxi HeLa Human cervical cancer cells HRP Horseradish peroxidase IAP Inhibitor of apoptosis Inhibitor 7 5,6,8-trichloro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid IP Immunoprecipitation IB Immunoblot kd Kinase dead/inactive MALDI-TOF Matrix assisted laser desorption ionization-time of flight mCi Millicurie MM cells Multiple myeloma cells MS Mass spectrometry Myc MASMEQKLISEEDLNN epitope

NF-KB Nuclear factor- KB NP-40 Nonidet P-40 PI' Residue C-terminally adjacent to the cleaved aspartic acid P2 Residue N-terminally adjacent to the cleaved aspartic acid PAGE Polyacrylamide gel electrophoresis PBST Phosphate-buffered saline + 0.1% Tween-20 PHK Phosphorylase kinase PD'K pathway Phosphatidylinositol-3 kinase pathway PML Promyelocytic leukemia gene PMSF Phenylmethylsulphonylfluoride PP2B Calcineurin B, phosphatase PTEN Phosphatase and tensin homolog deleted on chromosome 10 PTM Post-translational modifications pS Phosphorylated serine residue PT Phosphorylated threonine residue PVDF Polyvinylidene difluoride QR2 Quinone Reductase 2 or NQ02 RNAi RNA interference shRNA Small hairpin RNA

xxn SILAC Stable isotope labeling with amino acids in cell culture sPITS Sequence-based protein identifying targeting strategy TBB 4,5,6,7-Tetrabromo-lH-benzotriazole TBBz 4,5,6,7-tetrabromobenzimidazole TBCA Tetrabromocinnamic acid; IQA, 5-oxo-5,6-dihydroindolo-(l,2- a)quinazolin-7-yl)acetic acid TBST Tris-buffered saline + 0.05% Tween-20 U2-OS Human osteosarcoma cancer cells uL, mL Microliter, milliliter V, A Volts, Amps

xxiii 1

CHAPTER 1 INTRODUCTION1

1.1 The druggable kinome Protein kinases at distinct locations within cell signaling pathways can promote or repress changes in gene expression by the reversible phosphorylation of target proteins at specific residues. Perturbations in the regulation of signaling pathways by protein kinases can have dramatic effects on the control of cell growth, proliferation and apoptosis, resulting in a variety of human diseases, including cancer [1]. With the completion of the human genome sequence and the identification of all protein gene products, approximately 518 protein kinases emerged, comprising the human kinome [2]. Recently, prediction of the draggability of protein families determined that Ser/Thr protein kinases are amongst the most promising drug targets for future small molecule inhibitors, accounting for approximately 20% of the druggable genome [3, 4]. The successful development of Gleevec (Imatinib), a protein kinase inhibitor that targets the Bcr/Abl fusion protein in chronic mylogenous leukemia (CML), has provided a template for the development of future knowledge-based design of protein kinase inhibitors [5]. The development of other anti-cancer protein kinase inhibitors such as Sorafenib, which targets the Ser/Thr protein kinase Raf-1, as well as other tyrosine kinases, has also showed success in the treatment of advanced renal cell carcinoma [6]. Ongoing pre clinical studies have demonstrated the potential for targeting a wide variety of protein kinases for potential cancer therapies, including the protein kinase CK2.

1.2 Protein kinase CK2 CK2 (formerly known as Casein Kinase II) is a messenger-independent serine/threonine protein kinase that is ubiquitously distributed in eukaryotes, resides in a variety of cellular compartments, and phosphorylates, as well as interacts, with multiple cellular proteins [7]. CK2 is a constitutively active protein kinase implicated in cellular transformation and the development of tumorigenesis [8]. CK2 recognizes a consensus

1 This chapter has been published: Duncan JS and Litchfield DW. Too much of a good thing: The role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2. Biochimica et Biophysica Acta, Proteins and Proteomics. 2008 January; 784(l):33-47 2 sequence that includes Ser/Thr residues N-terminal to clusters of acidic residues, with a minimum requirement of S/T-X-X-D/E [8]. CK2 is most often present as a tetrameric complex that consists of two regulatory CK2(3 subunits and two catalytic subunits (a and/or a') in a homozygous or heterozygous composition [7]. In mammals, there are two isozymic forms of the catalytic subunit of CK2, designated CK2a (42-44 kDa) and CK2a' (38 kDa), that are the products of distinct genes localized to different chromosomes [8]. The catalytic subunits exhibit nearly 90% sequence identity in the N- terminal 330 amino acids and the amino acid sequences are highly conserved in higher eukaryotes with the exception of the unrelated (approximately 20 amino acids) carboxy terminus [7]. Although there is no known catalytic difference between CK2a and CK2a', there is evidence that CK2a and CK2a' exhibit functional specialization [9-12]. CK2a is phosphorylated by p34cdc2 at specific sites (Thr344, Thr360, Ser362, Ser370) located in its unique C-terminal domain during cell cycle progression while CK2a' is not phosphorylated [13]. The specificity of binding of cellular proteins such as HSP90, Pin-1, PP2A and CKIP-1 (CK2-interacting protein-1) with CK2cc and not CK2a' provide further evidence to support the notion that CK2a and CK2cc' have independent cellular functions [8, 14-18]. CK2 is a highly conserved enzyme implicated in cell proliferation, differentiation, and apoptosis and has been shown to be essential for survival in yeast [19-21]. Genetic studies in yeast have indicated that knockout of CK2A1 (a) and CK2A2 (a') catalytic subunits results in lethality and provided the first evidence for the functional specialization of CK2 a and a' [19, 22]. Distinct phenotypes in CK2a and CK2a' temperature-sensitive yeast mutants revealed that the loss of CK2a resulted in a defect in cell polarity, while loss of CK2a' resulted in cell cycle arrest [23, 24]. Studies in mammalian cells indicate that CK2 is required for the transition through G0/G1, Gl/S and G2/M [19, 25-27]. CK2 has been implicated in the G2/M transition through its association with the mitotic spindle, centrosomes, as well as cell cycle regulatory proteins, such as p34cdc2, cdc34 and topoisomerase II [12, 22, 28-30]. Knockout studies of CK2|3 in mice resulted in embryonic lethality, while knockout of CK2a' in mice, resulted in viable offspring that could be bred to homozygosity but were defective in 3 spermatogenesis [31, 32]. The results indicated that CK2(3 is essential for cell viability, while CK2cc can compensate for the loss of CK2a', but the compensation is not absolute [7, 31, 32]. Knockout studies of CK2a have not been published to date, presumably due to its essential role in cell viability.

1.3 CK2 promotes tumorigencsis Elevated CK2 activity has been associated with the malignant transformation of several tissues, as well as aggressive tumor behaviour [20]. Overexpression of CK2 has been documented in a number of cancers including, kidney [33], mammary gland [34], lung [35], head and neck [20] and prostate [36]. Targeted expression of CK2 in transgenic mouse models resulted in the transformation of T-cells and mammary gland epithelium leading to the development of tumorigenesis [34, 37]. In mouse models, targeted overexpression of both CK2 and either c-Myc [37] or Tal-1 [38] in T-cells, as well as the overexpression of CK2 in tumor suppressor p53 (-/-) mice [39], resulted in an increase in oncogenic activity and progression of tumorigenesis. A cooperative increase in oncogenic activity was also observed in BALB/c 3T3 fibroblasts co-expressing H-Ras and CK2a' [40]. The synergistic effect of CK2 in the promotion of tumorigenesis in cells overexpressing oncogenes, as well as cells lacking tumor suppressor activity, provided evidence for a crucial role for CK2 in the progression of oncogenesis. Perturbed CK2 expression has been implicated in the promotion of breast cancer, where overexpression of CK2 results in the dysregulation of key signaling pathways that control transcriptional regulation of mammary epithelium [34]. CK2 activity has been linked to Her-2/neu oncogene, which is overexpressed in 30% of breast cancers [41]. Increased CK2 activity is a key factor in the promotion of the Her-2/neu survival pathways leading to elevated growth rates [42]. Overall, the important role of CK2 in the development of tumorigenesis has become increasingly evident, as a number of tumor suppressor proteins, pro-apoptotic proteins, as well as oncogenes have been shown to be direct targets or protein interactors of CK2 (Figure 1-1). 4

Promotion of tumorigcnesls

Protection from ProtMtomo degradation cnput eltavaga •utctptibHtty 1 1 I 1 i HS1 PS-2 Connaxtn Bid Max 46.6

Figure 1-1 The molecular role ofCK2 in the progression of tumorigenesis. Flowchart depicting the three different mechanisms in which CK2 promotes tumorigenesis. Listed within each category are examples of selected protein targets that are affected by CK2 phosphorylation, where the J, represents promotion of the activity/stability while the -L represents degradation or decrease in activity. Enhanced CK2 activity results in the promotion of the degradation of tumor suppressor proteins (blue circle) and the prevention of degradation of oncogenes (red square) and pro-survival proteins facilitating the development of tumorigenesis. See text for more detailed discussion. 5

1.3.1. CK2 regulates the activity and stability of tumor suppressor proteins Tumor suppressor proteins function in the cell as gatekeepers that govern the progression of a cell through the cell cycle in addition to a key role in apoptosis [43]. Frequent loss of tumor suppressor activity in cells has been associated with multiple cancers implicating the critical role of these proteins in regulating the normal cellular events required for maintenance of cell viability [1]. The regulation of tumor suppressor activity and stability via the phosphorylation by CK2 has become an emerging paradigm. In fact, phosphorylation of tumor suppressors by CK2 has been shown to influence their functional activity, increasing or decreasing the affinity for targets [44], as well as protecting or promoting their degradation via the proteasome pathway [45, 46]. An essential tumor suppressor protein is p53, which has been implicated in the regulation of the cell cycle, differentiation and apoptosis [43]. The phosphorylation of p53 by CK2 at Ser392 has been observed in response to DNA damage resulting in increased DNA binding activity and transcriptional activity of p53 [47, 48]. A multi- protein FACT complex consisting of SSRP1 and hSPT16, as well as other proteins, form with CK2 upon DNA damage, which results in increased specificity for the phosphorylation of p53 [49]. Interestingly, SSRP1 has been shown to be phosphorylated by CK2, which negatively regulates transcription by preventing SSRP1 from binding to DNA, stalling transcription and replication following DNA damage [50]. This event has been proposed to lead to the formation of the FACT complex, which increases the phosphorylation of p53, resulting in attenuation of proliferation and increased apoptosis following DNA damage. It appears that p38 MAP kinase, another CK2-interacting protein, also has the ability to facilitate the phosphorylation of p53 at Ser392 in response to DNA damage [51]. Collectively, the phosphorylation of tumor suppressor p53 by CK2 in response to DNA damage has been shown to facilitate the activity of p53 leading to a cellular response. Another mechanism by which CK2 can influence the activity of tumor suppressor proteins is through the regulation of proteosome degradation susceptibility. An example of this is the control of protein stability by CK2 phosphorylation in the PML pathway. PML is a tumor suppressor that controls many pathways including growth suppression, 6 apoptosis and cellular senescence [52]. Loss of PML activity occurs in a large number of cancers and correlated with cancer susceptibility and tumor progression. CK2 phosphorylates PML at Ser517 which promotes the degradation of PML via the proteasome pathway protecting cells from apoptosis [46]. It has been proposed that enhanced CK2 activity observed in tumors will promote PML degradation resulting in the loss of its tumor suppressor activity and ultimately tumorigenesis. Furthermore, PML inactivation leads to tumor growth in a lung cancer mouse model system, while the inhibition of CK2 enhances PML tumor suppressor activity. The regulation of protein stability via phosphorylation by CK2 is also illustrated in the control of PTEN activity [45, 53, 54]. PTEN is a tumor suppressor involved in the regulation of cell survival and found to be frequently mutated in a number of cancers. The major of role of PTEN in tumor suppression is through the negative regulation of the phosphatidylinositol-3 kinase (PI3'K) pathway that promotes cell survival [55]. Survival signals are mediated by PTEN where it acts as a phosphodiesterase via the removal of a 3' phosphate group of phosphatidylinonsitol (3,4,5)-triphosphate with resulting inhibition of the phospho-Akt pathway. Phosphorylation of PTEN by CK2 has been reported to result in the stabilization of PTEN in its inactive form. Therefore, phosphorylation of PTEN by CK2 is thought to inhibit PTEN activity and perpetuate the PI3'K survival signal leading to oncogenesis. The examples of PML and PTEN illustrate how CK2 phosphorylation either facilitates the degradation of proteins or protects proteins from the proteasome degradation pathway. Overall, perturbations in this regulation can lead to loss of activity of the tumor suppressor resulting in the unregulated perpetuation of survival signals leading to the development of tumorigenesis. As described in the following section, the regulation of protein stability by CK2 phosphorylation has also been indicated in controlling the activity of a number of oncogenes that promote transformation and tumorigenesis.

1.3.2. CK2 promotes cell survival through the regulation of oncogenes The loss of regulation of proteins controlling cell growth is a major contributor in the development of cancer. Oncogenes are key proteins that promote the uncontrollable 7 growth of cells [1]. An understanding of the molecular mechanisms involved in the progression of tumorigenesis is essential to the development of anti-cancer therapeutics. Elevated CK2 activity has been associated with the promotion of a number of gene products that control cell proliferation, differentiation and apoptosis. Direct phosphorylation of oncogenic targets by CK2 has been shown to facilitate the cell survival signaling pathways leading to uncontrollable growth. A number of proto- oncogenic products including c-Myc [56], c-Myb [57] and c-Jun [58], as well as transcriptional activators NF-KB [42, 59, 60], |3-catenin [61] and Max [62] have been shown to be direct targets of CK2 where phosphorylation results in alteration of activity. The molecular mechanisms by which CK2 can influence multiple pathways that lead to promotion of cell survival are not fully understood. However, a number of signaling pathways have been extensively studied in which CK2 catalytic activity plays an essential role in the perpetuation of cell survival signaling. A detailed characterization of CK2- dependent pathways involved in the development of tumorigenesis will be pivotal to the development of cancer therapeutics.

1.4 CK2 -dependent pathways involved in development of cancer

1.4.1. NF-KB pathway

Uncontrolled regulation of the transcriptional-activator NF-KB has been implicated in the development of a variety of cancers via the loss of control of the cell

cycle and apoptosis [63]. The NF-KB transcriptional targets include cytokines, cyclin Dl,

anti-apoptotic proteins BcL-xl and cIAPs. Activation of NF-KB requires phosphorylation of IKB (inhibitor of KB) which promotes the degradation of IKB via the SCF-B-TrCP

proteasome pathway. The phosphorylation event required for NF-KB activation was determined to be CK2-dependent and soon after CK2 was shown to directly phosphorylate IKB [64]. Phosphorylation of IKB by CK2 facilitated the degradation of

IKB promoting transcription of NF-KB gene targets. CK2 has also been shown to

phosphorylate the NF-KB family member RelA/p65 following TNF-a stimulation, which

results in the enhanced DNA transactivation potential of NF-KB [65]. The interaction

between p65 and NF-KB in un-stimulated cells was shown to inhibit the phosphorylation 8 of p65 by CK2, resulting in reduced NF-KB activity. However, following TNF-a-signal- induced degradation of IKB the inhibition of CK2 by the p65-NF-KB complex was released, leading to enhanced NF-KB DNA binding activity and perpetuation of the pro- survival signal. It appears that CK2 functions in the NF-KB signaling pathway by promoting the degradation of IKB, as well as enhancing the DNA binding potential of

NF-KB through the phosphorylation of p65. CK2 has also been proposed as a potential gene product of NF-KB transactivation, implicating the presence of a positive feedback loop where CK2 drives its own expression [66]. Overall, the NF-KB pathway represents an intriguing route for perpetuating the CK2 pro-survival signaling by increasing the degradation of IKB, which promotes the expression of anti-apoptotic proteins, ultimately protecting the cell from apoptosis.

1.4.2. Wnt signaling pathway The Wnt signaling pathway plays an essential role in embryogenesis; however, when the pathway is activated in adult tissues it promotes transformation and tumorigenesis [67]. CK2 has been implicated in the reactivation of the Wnt pathway frequently observed in sporadic colon cancers. High levels of the transcription factor (3- catenin activated via the Wnt pathway results in the transcription of pro-survival signals such as c-Myc, c-Jun and cyclin Dl. Phosphorylation of |3-catenin by CK2 appears to be a pivotal event in the development of tumorigenesis by stabilizing |3-catenin and protecting it from proteasome degradation [68]. Prolonged activation of (3-catenin results in the perpetuation of pro-survival signals and oncogenesis. Furthermore, transgenic mouse models overexpressing CK2 or a dominant negative form of GSK3 results in the overexpression of |3-catenin leading to the development of mammary tumors [61]. In the presence of a Wnt signal, GSK3(3 is inactivated allowing CK2 phosphorylation of |3- catenin, which is proposed to promote the dissociation of P-catenin from its chaperones, APC and Axin. This dissociation event allows the translocation of (3-catenin to the nucleus leading to the activation of transcription of pro-survival signals. CK2 has also been shown to phosphorylate UBC3 and UBC3B, which are E2 ubiquitin-conjugating enzymes that interact with the F-box components of the B-TrCP 9

proteasome recognition complex [69]. The phosphorylation of UBC3 and UBC3B by CK2 was found to influence the recognition potential of the F-box for phosphorylated proteins, such as p-catenin, that are destined for proteasome degradation. The phosphorylation of UBC3 and UBC3B by CK2 provides further evidence for a role of CK2 in regulating protein stability within the Wnt pathway. By influencing the recognition potential of the proteasome machinery, CK2 could determine what proteins are destined for degradation, thus regulating their activity. The phosphorylation of the Wnt pathway components Dishevelled-2 (Dvl-2) and Dishevelled-3 (Dvl-3) by CK2 was discovered implicating another level of regulation of |3-catenin levels by CK2 [70, 71]. The inhibition of CK2 in Wnt-1-transfected cells was shown to result in the reduction of both Dvl protein levels and |3-catenin levels, indicating a potential regulatory role of Dvl activity by CK2. Dishevelled is thought to regulate the activity of GSK3|3 by blocking its ability to phosphorylate (3-catenin, which results in the degradation of P-catenin [67]. It appears that the phosphorylation of Dishevelled by CK2 may contribute to the perpetuation of the Wnt signal leading to an increase in |3-catenin levels and promotion of cell survival, however, further studies of the molecular consequence of the phosphorylation of Dishevelled by CK2 will be required. The tumor suppressor APC is another component of the Wnt pathway that contributes to the regulation of p-catenin levels that has been linked to CK2. APC has been shown to associate directly with CK2 and regulate its activity, where interaction appears to be cell cycle-specific, with the highest interaction during G2/M cell cycle [72]. The functional interaction between APC and CK2, as well as the ability of APC to inhibit CK2 activity, is thought to be a method by which APC regulates cell growth. Interestingly, the ability of APC to suppress CK2 activity is lost in C-terminal APC mutants, indicating that full-length APC is required for the regulation of CK2 activity. Loss of the C-terminal of APC through truncation mutations is frequently found in sporadic colon cancer, implicating that the loss of APC regulation of CK2 activity may be an important event in the development of tumorigenesis. In other studies, APC cellular localization was shown to be regulated by CK2 [73]. Studies revealed that p38 MAPK was capable of directly associating with and modulating CK2 and PKA activity, promoting the block of nuclear import of APC. Disruption of nuclear import of APC to 10 the nucleus by CK2 resulted in increased levels of (3-catenin, promoting the transcription of pro-survival signals and progression of tumorigenesis. CK2 appears to be essential for the perpetuation of the Wnt signaling pathway, through the regulation of multiple pathway components. The consequence of elevated CK2 in cancer cells is the maintenance of high levels of |3-catenin in cells, which promotes tumorigenesis through the persistent transcription of pro-survival genes. Regulation of (3-catenin levels by CK2 has been shown via a number of different pathways, where increased CK2 activity resulted in increased transcription of (3-catenin gene targets. Expression of a |3-catenin transcription target survivin has recently been linked to increased CK2 activity and tumorigenesis [74]. Survivin is a member of the inhibitor-of-apoptosis family, IAP, involved in apoptosis and cell cycle progression. Survivin acts as an anti-apoptotic protein where it inactivates caspases and stabilizes inhibitors-of-apoptosis. Survivin expression normally occurs during embryonic development; however, expression of survivin has been detected in a number of cancers implicating survivin in tumorigenesis. Interestingly, elevated CK2 activity was shown to result in the upregulation of survivin expression through the (3-catenin/Tcf pathway. The upregulation of survivin by enhanced CK2 activity leads to the promotion of cell survival and tumorigenesis, reiterating the attractiveness of CK2 as an anti-cancer therapeutic target. Perturbed regulation of the proto-oncogene c-Myc has been implicated in the development of a number cancers [75]. The nuclear transcription factor c-Myc is essential for mammalian development and is upregulated in many malignancies. c-Myc transcription can be activated by the (3-catenin/Tcf/LEF transcription factors of the Wnt pathway, resulting in the loss of regulation of cellular proliferation [76]. Overexpression of CK2a and c-Myc in transgenic mice lymphocytes results in the development of lymphoid leukemias illustrating a strong biological synergy between CK2 activity and c- Myc transcriptional activation [37]. A functional interaction between CK2 and c-Myc was investigated to determine the molecular mechanism for the biological synergy seen in the overexpression studies [56]. CK2 was found to associate with and phosphorylate c-Myc on the C-terminal PEST domain. The phosphorylation of c-Myc prevents its degradation via the proteasome pathway, allowing it to propagate its transcriptional-activator signal 11 and promote expression of survival genes. Therefore, elevated CK2 activity would promote increased levels of the stable c-Myc oncogene, which would promote cellular proliferation and contribute to the development of cancer.

1.4.3. PB'K pathway The PD'K pathway controls cell survival through a number of converging signaling pathways [77]. Phosphorylation of Akt promotes the cell survival signal through the NF-KB signaling pathway, direct regulation of caspase activity, and inactivation of the pro-apoptotic protein BAD. CK2 has been shown to phosphorylate Aktl at Ser 129 in vivo which promotes cell survival by generating a constitutively-active form of Akt [78]. In this situation, the pro-survival signal of the PB'K pathway is continually active when Akt is phosphorylated by CK2 leading to the development of tumorigenesis. As noted earlier, CK2 has previously been linked to the regulation of PTEN protein stability, which controls the activation of Akt through its phosphatase activity of PI3K [45]. It appears that CK2 is functioning in multiple points within the PD'K pathway and has a key role in promoting the survival signal. CK2 can regulate Akt activity indirectly by controlling the activity of PTEN through proteasome-dependent phosphorylation, as well as activate Akt by direct phosphorylation. Taken together, elevated CK2 activity can facilitate the PB'K cell survival signaling pathway through the constitutive activation of Akt, which promotes cell survival and progression of tumorigenesis (Figure 1-2).

1.5 An emerging anti-apoptotic role for CK2 The involvement of CK2 in the regulation and perpetuation of a number of cell survival signaling pathways and its ability to transform and promote tumorigenesis raises the possibility of an anti-apoptotic role for CK2. The activation of a number of anti- apoptotic proteins via the Wnt, NF-KB or PB'K pathways by elevated CK2 activity has promoted a number of studies investigating the direct role of CK2 in the apoptotic pathways. Elucidation of the molecular mechanisms by which CK2 functions in apoptosis will be beneficial in the generation of knowledge-based anti-cancer therapeutics. CK2 12 has been implicated in direct regulation of receptor-mediated apoptosis [79], as well as intracellular apoptosis through the DNA damage-induced apoptosis pathways [80].

Elevated CK2 levels Inhibition of CK2

B RTK RTK

PI3 kinase

|—(^TEN)

^fcaspase) (BM) I Survival Apoptosis

Figure 1-2. The role ofCK2 in the perpetuation of the PI3'K signaling pathway. (A) Overexpression of CK2 in the PD'K pathway results in the inactivation of the tumor suppressor PTEN and the constitutive activation of the oncogene Akt, promoting cell survival. Phosphorylation of PTEN by CK2 promotes the stabilization of PTEN in its inactive form, thus preventing it from inhibiting the PD'K survival signal. Phosphorylation of Akt by CK2 results in the generation of a constitutively active form of Akt, which inhibits the progression of apoptosis via the inhibitory phosphorylation of pro- apoptotic proteins. (B) Inhibition of CK2 results in the loss of phosphorylation of PTEN and Akt, thus inhibiting the PD'K survival pathway by active PTEN. 13

Interestingly, inhibition of CK2 in various cancer cells has been shown to sensitize cells to receptor-mediated [81] and intracellular apoptosis [82] reiterating the essential role of CK2 in maintenance of cell survival. Sensitization of cells to apoptosis following CK2 inhibition has been studied extensively with hopes of utilizing CK2 inhibitors in conjunction with chemotherapy or radiation-therapy in cancer treatments. Increasing evidence also suggests that CK2 has a general anti-apoptotic role in cells via the protection of pro-apoptotic proteins from caspase-mediated cleavage [7]. CK2 phosphorylates pro-apoptotic proteins in close proximity to the caspase mediated cleavage site, protecting the cells from caspase-dependent degradation and ultimately from entering apoptosis [83]. The ability of CK2 to sensitize and directly inhibit apoptosis in cancer cells provides evidence that CK2 acts directly on the apoptotic signaling pathway through regulation of transcription of anti-apoptotic proteins, as well as direct regulation of caspase activity by preventing caspase cleavage of targets.

1.5.1. Evidence of sensitization of cells to apoptosis following inhibition ofCK2 Downregulation of CK2 activity by a number of strategies in cancer cells sensitizes the cells to apoptosis. Knockdown of CK2ot using RNAi or overexpression of kinase-inactive CK2cc resulted in an increase in TRAIL-induced sensitivity in Rhadomyosarcoma and HT29 colon cancer cells [79, 81]. Sensitization to apoptosis was also observed following treatment with CK2 inhibitors, as shown by an increase in TRAIL-induced death signaling complex (DISC) formation, enhanced caspase 8 and Bid cleavage, as well as the downregulation of the anti-apoptotic proteins xIAP and c-IAP. Knockdown studies of CK2 using RNAi were also performed to address the role of CK2 in response to ionizing radiation (IR) damage in HeLa cell lines [80]. CK2 was found to negatively regulate apoptosis in IR damage and affected the cell cycle progression. It was also observed that androgen-insensitive PC-3 and androgen-sensitive ALVA-41 prostate cells are sensitized to TRAIL-induced apoptosis following inhibition of CK2 [84]. The overexpression of CK2a resulted in the protection of prostate cells from TRAIL/Apo2L- mediated apoptosis, providing further evidence for a role of CK2 in regulation of receptor-mediated apoptosis [85]. A recent study investigating the molecular mechanism of the sensitization of cells to apoptosis following treatment with chemotherapeutic 14 compounds Resveratol and EGCG found that CK2 was a critical component in the signaling event [86]. A similar study was performed investigating the role of CK2 in the induction of apoptosis caused by treatment of leukemia cells with the chemotherapeutic agent 6-TG [82]. Knockdown of CK2 using RNAi in conjunction with 6-TG treatment was performed in HeLa and HCT116 cells to address the molecular mechanism by which CK2 functions in sensitization of cells to chemotherapy. The results indicated that 6-TG- induced DNA damage requires the activity of CK2 for inhibiting apoptosis and controlling caspase activity. Another interesting study was performed to elucidate the ability of CK2 to promote chemical resistance to various chemotherapeutics and to address whether inhibition of CK2 can sensitize resistant cells to apoptosis [87]. Inhibition of CK2 resulted in the sensitization of CEM cell lines to apoptosis and promoted the increased uptake of therapeutic drugs. The ability of CK2 to sensitize resistant and non-resistant cells to apoptosis raises the possibility of using CK2 inhibitors in conjunction with chemotherapeutics to treat various cancers. If specific cancers exhibit resistance to conventional chemotherapy treatment, CK2 inhibitors could be employed to sensitize the tumors to apoptosis.

1.5.2. Regulation ofapoptotic machinery by CK2 Induction of apoptosis in cells following downregulation of CK2 activity has provided further evidence of an anti-apoptotic role for CK2 [88]. Downregulation of CK2 activity by means of antisense [89], RNAi [87], overexpression of kinase inactive CK2 [90, 91] or chemical inhibition [92] have shown the induction of apoptosis in cancer cells, reiterating the importance of CK2 in maintenance of cell survival. A number of investigations to unravel the molecular mechanism by which CK2-inhibited cells undergo apoptosis have been undertaken. Interestingly, a strong similarity exists between the caspase degradation recognition sequence and the CK2 consensus motif for phosphorylation, signifying that CK2 may be involved in protection of a wide variety of caspase targets. CK2 phosphorylation of the caspase targets BID [93], Max [94], HS1 [95], PS-2 [96], Connexin 45.6 [97] and PTEN [98] at a position proximal to the caspase recognition site results in protection from caspase cleavage. Focusing on PTEN, a key tumor 15 suppressor in the PB'K pathway, it was demonstrated that phosphorylation by CK2 at the caspase 3 cleavage site, protected PTEN from caspase cleavage and promoted stability [98]. Interestingly, PTEN protein stability has been shown to be influenced by CK2 phosphorylation where it regulates its activity by preventing proteasome degradation. So it appears that CK2 is able to regulate PTEN activity and protein stability through overlapping protection from both the proteasome and caspase degradation pathways. Promotion and repression of the activity of apoptotic proteins by CK2 phosphorylation has also been documented, implicating a direct role of CK2 in protection of cells from apoptosis. The phosphorylation of procaspase-2 by CK2 has been shown to prevent the activation of caspase activity by inhibiting the dimerization of procaspase-2 [99]. This regulation of caspase activity by preventing dimerization illustrates another mechanism by which CK2 can inhibit apoptosis. The activity of ARC, a caspase- inhibiting protein also requires phosphorylation by CK2 in order for it to inhibit the activity of caspase 8 [100]. It appears that CK2 activity is involved in the direct regulation of the caspase 8 pathway, where it can control caspase 8 activity through the phosphorylation of ARC or by preventing the ability of caspase 8 to cleave its target Bid. The ability of CK2 to directly regulate components of the apoptotic machinery provides further evidence for an emerging role of CK2 in the protection of cells from apoptosis. A comprehensive evaluation of the molecular mechanisms by which CK2 functions in apoptosis using strategies to inhibit CK2 in cells will be critical for the identification of CK2 targets, as well as the determination of the druggability of CK2 in anti-cancer therapeutics.

1.6 Exploitation of strategies to inhibit CK2 function Mounting evidence suggests that CK2 has a general anti-apoptotic role in cells via the regulation of multiple pro-apoptotic transcription factors, the direct regulation of tumor suppressor and oncogene protein stability, as well as the emerging protection of pro-apoptotic proteins from caspase-mediated cleavage. Overexpression studies of kinase inactive CK2a and CK2a', as well as knockdown studies of CK2ct and CK2a' using anti-sense or RNA interference and chemical inhibition were shown to result in an attenuation of proliferation and induction of apoptosis in various cell lines, reiterating the 16 importance of CK2 in the regulation of cell survival [87-90]. Consequently, CK2 has emerged as a potential therapeutic target, and strategies to inhibit CK2 have been ongoing in pre-clinical trials. Limitations in kinase-inactive dominant-negative studies as well as knockdown studies of CK2 have become evident, due to long half-life, cellular localization and high expression of CK2 in cells [7, 101]. Overexpression of kinase-inactive CK2 will always have the limitation of high expression of endogenous CK2 within the cells, which can confound or mask the effect of the loss of CK2 kinase activity. The use of RNA interference to selectively downregulate the endogenous CK2, but not the kinase-inactive mutant, may provide opportunities to investigate the consequence of the loss of CK2 kinase activity. However, with the development of new selective inhibitors for CK2, kinase-inactive studies may become secondary tools to study CK2 function. The limitation of downregulation studies was illustrated in a study performed to investigate CK2 as an oncogenic target [101]. Using anti-sense oligodeoxynucleotide strategies and RNAi the investigators found that the biological inhibition strategies differed in their effectiveness to reduce CK2 expression levels. Anti-sense oligodeoxynucleotide strategies were shown to inhibit CK2 activity by 50% but failed to reduce CK2 levels in cells, whereas knockdown of CK2 using RNAi inhibited activity and slightly reduced CK2 expression levels. However, previous studies utilizing anti-sense or RNAi strategies have shown sufficient reduction in CK2 levels in various cell lines indicating that there may be unique cell line-specific properties that influence the effectiveness of the downregulation strategies [89]. The conflicting effectiveness of knockdown studies could in part be due to enhanced stability or altered expression levels of CK2 in various cell types. The existence of sequestered CK2 protein complexes or highly ordered CK2 holoenzyme structures that promote stability could prevent a sufficient knockdown of CK2 [101, 102]. Utilization of RNAi or anti-sense oligodeoxynucleotide strategies to inhibit CK2 could prove to be inefficient, requiring continual treatment of cells with knockdown strategies in order to achieve CK2 depletion. The inability to remove all CK2 from knockdown cells will be problematic in addressing the mechanism by which CK2 functions in cell survival where a minimal amount of CK2 may be sufficient to maintain function. Therefore, the pharmaceutical inhibition of CK2 has become a promising tool in the study 17 of CK2 in various cell survival pathways. Chemical inhibitors would be able to presumably penetrate and inhibit all CK2 in cells, allowing the investigation of the effect of CK2 depletion on cellular decisions of life and death.

1.7 Knowledge-based design of CK2 inhibitors A number of different classes of chemical compounds have been investigated as ATP competitive inhibitors of CK2 that differ in their efficacy and specificity (Table 1-1). There are four major classes of CK2 inhibitors: Quercetin, which includes Apigenin; Emodin, TBB (4,5,6,7-Tetrabromo-lH-benzotriazole) and its derivatives, and IQA (5- oxo-5,6-dihydroindolo-(l,2-a)quinazolin-7-yl)acetic acid) [103]. Apigenin and Emodin have been shown to have limited selectivity for CK2, whereas TBB and IQA show high specificity for CK2 representing the first promising compounds for the study of CK2 function. Derivatives of TBB, mainly TBBz (4,5,6,7-tetrabromobenzimidazole), DMAT (2-Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole) and TBCA (Tetrabromocinnamic acid) have been developed in an attempt to increase the selectivity of inhibitors towards CK2 [104-107]. Inhibition of other kinases by TBB, DMAT and TBCA was evaluated revealing that protein kinase DYRKla was inhibited by both TBB and DMAT, indicating that the inhibitors are highly selective for CK2 but still have potential off-targets [105]. Interestingly, inhibition of DYRKla was not observed with TBCA, implicating TBCA as the most selective inhibitor of the TBB derivatives. A number of new CK2 inhibitors that deviate structurally from the TBB derivatives have been identified through the screening of a variety of chemical libraries. The discovery of IQA from a virtual screening of the Novartis collection of compounds, revealed that IQA was a highly selective inhibitor for CK2, where it had no inhibitory effect on DYRKla or any other protein kinase from a panel of 30 protein kinases tested [108]. Ellagic acid, which is a naturally occurring tannic acid derivative, was identified as a CK2 inhibitor through a virtual screening application and represents the inhibitor with the lowest Kj for CK2 [109]. Ellagic acid was shown to inhibit CK2 with high specificity indicating that other compounds structurally unique from the TBB derivatives may be useful in the study of CK2. 18

A large number of highly specific chemical inhibitors of CK2 have been identified; however, the molecular mechanism of action of these compounds has not been systematically characterized. In particular, a detailed examination of the physiological effects of CK2 inhibitors in cells will be required to address the specificity of CK2 inhibitors in vivo, as well as their potential use as anti-cancer therapeutic drugs.

Table 1-1. A comparison of the efficacy ofCK2 inhibitors.

CK2 inhibitor IC50=(xM Kj=(xM Reference Apigenin 0.8 0.74 [108, 128] Emodin 2.0 1.85 [108] DRB 13.0 4.50 [108,138] TBB 0.50 0.40 [103] TBBz (TBI) 0.50 0.70 [88, 92] DMAT 0.14 0.04 [104] TBCA 0.11 0.077 [105] IQA 0.39 0.17 [108] Ellagic Acid 0.04 0.02 [109] Inhibitor 7 0.30 0.06 [129]

1.8 Characterization of CK2 inhibitors in vivo To support the emerging role of CK2 in the protection of cells from apoptosis, a number of studies have been performed to determine the effect of CK2 inhibitors on cell viability. Treatment of a variety of cancer cells with cell-permeable CK2 inhibitors including DRB [110], Emodin [111], TBB [106], TBBz [88], DMAT [104], IQA [108] and TBCA [105] resulted in the induction of apoptosis through the activation of caspases. Furthermore, a recent example involved the use of CK2 inhibitors in combination with Imatinib (Gleevec) to treat cells that express the Bcr/Abl oncogene [112]. Treatment of the PLC1 cell line established from the PI90 Bcr/Abl transgenic mouse with Imatinib in combination with DMAT resulted in a cooperative reduction in cell viability. Another study utilizing CK2 inhibitors to investigate the anti-tumorigenic role of PML, found that treatment of mice expressing Colo320DM cells with emodin inhibited tumor growth [46]. Inhibition of CK2 has also been shown to lead to the downregulation of the AR- 19 dependent transcription and reduced AR protein levels in prostate cancer cells [113]. AR pathways are essential for the development and progression of prostate cancer, therefore, inhibition of this pathway by CK2 inhibitors could provide a model for the development of new anti-cancer therapies for this form of cancer. Due to the striking anti-tumor effect of CK2 inhibitors, many studies have been undertaken to evaluate and characterize the effect of various inhibitors on the viability of cancer cells. Interestingly, treatment of HeLa cells with 25 ^M TBB or TBBz resulted in differences in cell viability, indicating that different inhibitors can have unique cellular effects [88]. Cells treated with TBBz resulted in the induction of apoptosis, while TBB failed to induce an apoptotic response. However, both drugs are capable of inhibiting CK2 in vitro with similar Kj values; indicating a potential difference in the drug permeability as an explanation of the differences in the inhibitors phenotype. Furthermore, TBBz has demonstrated isoform specificity compared with TBB, where it effectively inhibits the CK2 holoenzyme in yeast but is ineffective at inhibiting the free CK2a'. This raises the possibility for inhibitor selectivity between individual catalytic subunits of CK2 [114]. A greater difference in the ability of TBBz to distinguish between human CK2a and CK2a' was also observed compared to TBB, making it a better candidate for isozyme-specific studies [88]. The observed differences in the ability of TBB and TBBz to induce apoptosis in cells may be attributed to the fact that TBBz is predominately in a neutral form at physiological pH, while TBB exits exclusively as the mono-anion. The chemical properties of these CK2 inhibitors may be the primary determinant of their ability to become cell-permeable under physiological conditions, as well as their ability to target and inhibit CK2 in vivo. An evaluation of the physiological effects of the current CK2 inhibitors is necessary to address CK2 inhibitor pharmokinetics and drug stability within cells. In order to effectively evaluate the differences in cellular responses of various CK2 inhibitors, systematic studies must be performed to determine the off target effects of the drugs. In this respect as described earlier, a number of the CK2 inhibitors have been shown to inhibit other kinases, including DYRKla, which is a protein involved in a number of cellular processes, including apoptosis [92]. Inhibitors, such as TBB, were demonstrated to inhibit other protein kinases with less potency than CK2 including CDK2 20 and GSK3J3. Potential off-target inhibition of GSK3|3 by CK2 inhibitors, could influence the Wnt pathway signal leading to changes in the transcription of pro-survival signals. Due to the involvement of both CK2 and GSK3|3 in the regulation of the Wnt pathway, determining whether the cellular effect is CK2-dependent would require further validation studies [103, 108]. The standard assay for CK2 inhibitor specificity is to screen the inhibitor against a panel of representative kinases from each kinase family and test for diminished kinase activity. The limitation of using these kinase panels to test for specificity is that it is not comprehensive, raising the concern that their may be an inhibitor-sensitive kinase that was not screened. Importantly, the assays are not performed under physiological conditions where other kinases and ATP-binding proteins can compete for the inhibitor. Other ATP-binding proteins such as heat shock proteins, which may be influenced by the presence of the inhibitor, have been overlooked by focusing on a select group of ATP-binding proteins. Unbiased experiments utilizing chemo-proteomic approaches to identify the mechanism of action of CK2 inhibitors would be extremely beneficial. A chemo-proteomic approach was successfully employed in the identification of inhibitor targets of the malaria drug, quinoline, which identified ALDH1 and QR2 as selective targets for this drug [115]. A proteomic screen of TBB protein interactors could provide critical information on the specificity, as well as the molecular mechanism of action of the current CK2 inhibitors.

1.9 Identification of CK2 targets utilizing chemical inhibitors The number of putative CK2 substrates is continually growing with greater than 300 substrates to date; however, the number of validated in vivo targets remains a small subset of the total [116]. The development of highly-specific inhibitors has provided a tool to investigate the phosphorylation of various substrates, as well as to validate the targets as bona fide CK2 substrates. The use of CK2 inhibitors to identify CK2 targets has been investigated using a number of experimental strategies, including; observing a reduction in 32P incorporation in putative CK2 targets, inhibiting CK2 site-specific phospho-antibody recognition and preventing gel shift attributed to phosphorylation. A number of interesting putative CK2 targets have been identified utilizing in vivo 32P labeling of cells including the F-actin capping (CPa) protein, which was found to be 21 phosphorylated at Ser9 both in vitro and in vivo by CK2 [117]. Treatment of P labeled cells with 75 uM TBB resulted in reduction in phosphorylation of CPa implicating it as an in vivo putative CK2 target. The implications of CK2 phosphorylation of CPa have yet to be addressed but raise the prospect that CK2 functions in regulation of cell morphology [118]. The CK2-dependent phosphorylation of the cell cycle regulatory protein Geminin was also assessed through the use of CK2 inhibitors [119]. The phosphorylation of Geminin was reduced dramatically in 32P-labeled HeLa cells treated with 25 fxM TBB, implicating Geminin as a putative CK2 target. The hematopoietic lineage cell-specific protein 1 (HS1), which is involved in B cell apoptosis has been identified as a possible CK2 target [95]. Treatment of Jurkat cells with 40 \xM TBB resulted in the loss of phosphorylation as indicated by the loss of the upper phosphorylated band. The determination of phosphorylation using 32P labeling and phosphorylation shifts on a gel in conjunction with CK2 inhibitors are both effective at indicating a putative CK2 phosphorylation event, however, more specific methods are required to validate that the phosphorylation is indeed CK2 dependent. The use of phospho-specific antibodies recognizing CK2 phosphorylation sites in conjunction with CK2 inhibitors has provided opportunities to validate putative CK2 sites. An essential kinase in the PB'K signaling pathway, Akt, was found to be phosphorylated by CK2 at Serl29 and the phosphorylation could be reduced upon treatment with 50 uM TBB as indicated by reduction in phospho-Serl29 antibody signal [78, 87]. As noted earlier, phosphorylation of Akt by CK2 has been proposed to produce a constitutively active phospho-Akt which promotes cell survival and the development of tumorigenesis. The use of CK2-specific phospho-antibodies is also well illustrated in the study addressing the effect of CK2 phosphorylation on PML stability and its impact on tumorigenesis [46]. A CK2 phosphorylation site was identified in vitro in the PML protein at Ser517, which when mutated resulted in the loss of phosphorylation by CK2. A phospho-specific antibody was developed against Ser517 and was used to determine whether changes in CK2 activity in cells corresponded to changes in phosphorylation of Ser517. UV-radiated NIH-3T3 cells were treated with TBB and a decrease in phosphorylation of PML was observed as indicated by the loss of phospho-Ser517 antibody reactivity. Another example of the use of CK2 inhibitors was the treatment of 22

R-CEM cells with 20 \iM TBB to investigate the phosphorylation state of the apoptotic protein BAD [87, 120, 121]. Phospho-specific antibodies recognizing the phospho-Thr 117 CK2 phosphorylation site was reduced in the presence of TBB implicating BAD as a target of CK2 in cells. An investigation of the effect of elevated CK2 activity in multiple myeloma (MM) cells was assessed via the evaluation of phosphorylation changes upon CK2 inhibition [122]. The potential phosphorylation of IKB by CK2 at Ser32-36 was examined using phospho-specific antibodies in MM cells following TNF-cc stimulation. Treatment of cells with the CK2 inhibitor K27 resulted in decreased phosphorylation of IKB as detected by loss of reactivity of phospho-antibodies, implicating that CK2 is either directly or indirectly involved in the phosphorylation of Ser32-33 of IKB. The development of phospho-specific antibodies directed against CK2 sites and the use of CK2-specific inhibitors provides a strong experimental approach to validate putative CK2 targets as bona fide targets.

1.10 Validation of CK2 targets "identification of bona fide targets" A number of experimental strategies have been designed to identify CK2 substrates, which has led to the discovery of many potential CK2 targets. However, the number of validated bona fide CK2 targets is still limited relative to the number of putative in vitro and in vivo targets. In order to fall under the category of a bona fide CK2 target, the target must conform to two criteria; 1) the phosphorylation event must occur in vivo, and 2) the phosphorylation event must change with CK2 activity [7]. The use of CK2-specific inhibitors to identify CK2 targets has been quite effective at providing information on the phosphorylation state of the target protein but has its limitations. As illustrated by the examples cited earlier, immuno-precipitation of a putative CK2 target from 32P-labeled cells treated with high levels of CK2 inhibitors has proven to be effective at preventing the incorporation of 32P into the CK2 target protein. However, the consequence of treating cells with high doses of CK2 inhibitors on other essential cellular processes has not been extensively investigated. The potential for inhibitor off-target drug effects raises the concern that the decrease in phosphorylation of the putative CK2 target is due to inhibition of other ATP binding proteins or the disruption of essential cellular events that regulate viability. Apoptosis and attenuation of 23 proliferation of cells treated with CK2 inhibitors at concentrations as low as 5 [xM has been observed, reiterating the sensitivity of cells to CK2 inhibitors [104]. The combination of using CK2 inhibitors with other strategies, such as RNAi to specifically downregulate CK2 levels may provide a method for validating CK2 targets. The comparison of off-target phosphorylation effects between inhibitor and RNAi-treated cells could provide information on the specificity of the inhibitor, as well as validate it as a CK2 target. One such study was carried out looking at the phosphorylation and regulation of a G-protein Coupled Receptor by CK2 [123]. Inhibition of CK2 via inhibitor-treatment and RNAi-mediated knockdown both showed that CK2 contributes to the phosphorylation of M3-muscarnic receptor, which can determine specific signaling events. Interestingly, the comparison of in vitro phospho-peptide maps with in vivo phospho-peptide maps from both RNAi and inhibitor-treated cells showed changes in the same subset of phospho-peptides, providing strong evidence that M3-muscarininc receptor was phosphorylated by CK2. The combination of a variety of strategies to interfere with CK2 function can provide evidence that a target or phenotype is CK2- dependent. However, the change will require rescuing by the re-introduction of active CK2 to validate that the effect is directly associated with CK2. The development of highly-specific CK2 inhibitors has provided a strong platform for identifying CK2 targets but still has the limitation that they are not 100% specific as indicated by their ability to inhibit other kinases in the panel screens. ATP-binding site similarities between most protein kinases has been the critical limitation in the development of specific protein kinase inhibitors. To address the limited specificity of ATP-competitive inhibitors, a study successfully used engineered drug-resistant mutants to look at the specificity of the Stress Activated Protein Kinase /p38 inhibitor SB 203580 [124]. Therefore, the use of rescue experiments utilizing inhibitor-resistant CK2 mutants could provide a validation method in the identification of bona fide CK2 targets. The ability to employ inhibitor-resistant mutants to rescue changes in phosphorylation observed following treatment with CK2 inhibitors will provide substantial evidence that the change is due to the inhibition of CK2 and not an off-target effect of the inhibitor. CK2 inhibitor-resistant mutants have been developed through detailed studies of the crystal structure of CK2 and the interactions of specific hydrophobic residues within the 24 catalytic binding pocket with ATP. The unique properties of the ATP binding pocket of CK2, mainly the presence of large bulky hydrophobic residues, allow for the generation of inhibitor-resistant mutants [103]. Mutation of the unique hydrophobic residues that make up the hydrogen bonding network with the CK2 inhibitors, results in a CK2 mutant that is structurally more similar to other families of protein kinases. The mutant kinase is able to bind to ATP, but will bind through a network of interactions similar to that of other kinases, allowing it to remain catalytically active but resistant to the inhibitors. The mutation of the bulky hydrophobic residues (Val 66 -> Ala and He 174 -> Ala) results in the generation of a catalytically active CK2 that shows resistance to CK2 inhibitors TBB, DMAT, IQA and TBBz [88, 103]. An in vitro study looking at the sensitivity of inhibitor-resistant CK2ctR [(CK2a-(V66A/I174A)] to treatment with TBB or TBBz found that free CK2aR was more resistant to TBBz than TBB, indicating that some CK2 inhibitors may be more useful in rescue experiments utilizing the inhibitor-resistant mutants. The proof of principle that the inhibitor-resistant mutants were capable of rescuing the inhibition of CK2 by inhibitors was demonstrated in an experiment using cell lysates expressing the resistant CK2 mutants [125]. CK2 kinase activity was assessed by the phosphorylation of a synthetic peptide substrate in cell lysates expressing either wild type CK2aor V66A/I174A CK2a-resistant mutants treated with 5 uM TBB. The kinase activity of the CK2a inhibitor-resistant mutants was unaffected by treatment with TBB, illustrating that the inhibitor-resistant mutants will be useful in performing rescue experiments to validate CK2 targets explored using CK2 inhibitors. An investigation of the consequence of high CK2 activity on the cell survival of multiple myeloma (MM) cells revealed that treatment with TBB resulted in an induction of apoptosis that could be rescued by the expression of inhibitor-resistant V66A/I174A CK2a mutants [122]. The ability to inhibit the induction of apoptosis in MM cells treated with TBB using inhibitor- resistant mutants strongly suggests that CK2 is an essential protein kinase required for the regulation of cell viability. Inhibitor-resistant mutants have also been used to validate the role of CK2 in chromosomal DNA strand break repair and the maintenance of genomic integrity [126]. Treatment of EM9-XH cells with TBB prevented the assembly of H2O2- induced XRCC1 foci, however, this was not observed in the CK2ct V66A mutant cells. 25

This experiment demonstrates that CK2 has a direct role in DNA strand break repair, where CK2 activity is essential in the DNA single-strand break repair mechanism by enzyme XRCC1. The generation of engineered inhibitor-resistant CK2a (V66A/I174A) mutants will allow the systematic evaluation and validation of the many previously known putative CK2 targets, as well as aid in the discovery of novel CK2 targets. The development of CK2a'R (V67A/I175A) inhibitor-resistant mutants will be of interest in the facilitation of the discovery of novel CK2 substrates and will also provide information on the isoform-specific function of the catalytic subunits of CK2. A large number of putative CK2a substrates have been identified compared to the limited number of identified CK2a' targets, reiterating the importance of isoform-specific function. A recent study investigating the tumor suppressor NKX3.1 as a CK2 target in prostate cancer cells revealed that it could be phosphorylated by free CK2cc' but not CK2a or CK2a' in complex with the holoenzyme [127]. This study provides evidence for an isoform-specific function of CK2a' via the phosphorylation and regulation of NKX3.1. The use of CK2aR and CK2a'R to validate inhibitor-dependent phenotypes and to identify isoform-specific CK2 targets will provide information on the mechanism of action of CK2 inhibitors in cancer cells, as well as provide information on the role that CK2cc/a' play in the protection of cells from apoptosis.

1.11 Scope of Thesis Protein kinase CK2 plays an essential role in the development of tumorigenesis and has emerged as a promising drug target for anti-cancer therapeutics. Elevated CK2 levels have been implicated in the perpetuation of a number of key survival signaling pathways, thereby promoting the development of cancer via the protection of cells from apoptosis. Consequently, a number of strategies have been employed to interfere with CK2 activity, including the development of pharmaceutical CK2 inhibitors. The use of CK2 inhibitors to study CK2 function has provided critical information on the role of CK2 in the sensitization of tumor cells to conventional cancer treatments, as well as facilitated the identification of a large number of putative CK2 substrates. However, a detailed evaluation of the molecular mechanism of drug action, as well as inhibitor specificity of these compounds in cells has not been systematically explored. Therefore, 26 insight into the molecular mechanism by which CK2 inhibitors exhibit their anti-cancer properties is critical for understanding the functional role of CK2 in tumorigenesis. Chapter 1, focused on published evidence highlighting the molecular mechanisms by which CK2 functions in the promotion of tumorigenesis, as well as reviewed current strategies being used to inhibit CK2. The work presented in this thesis will extend the knowledge-base in the area of the role of CK2 in the development of cancer, specifically highlighting global techniques to evaluate protein kinase inhibitor specificity, as well as the molecular mechanisms by which CK2 protects cells from apoptosis promoting tumorigenesis, In Chapter 2, an unbiased evaluation of TBB and related CK2 inhibitors was carried out using chemo-proteomics to investigate the potential of the inhibitors as anti cancer therapeutics. A detailed study of the specificity of the CK2 inhibitors TBB, TBBz and DMAT for CK2, as well as the cellular effects associated with each inhibitor was carried out. Differences between inhibitors were observed with respect to the induction of apoptosis following treatment of HeLa cells with TBB, TBBz or DMAT, indicating that the inhibitors have unique biological properties, as well as revealed the potential for off-target effects. Therefore, rescue experiments utilizing inhibitor-resistant CK2 mutants were employed to investigate the specificity of the inhibitors, in particular, whether the apoptosis associated with drug treatment was CK2 dependent. The inability to rescue TBBz- and DMAT-induced apoptosis with the resistant mutants prompted a systematic characterization of inhibitor protein targets utilizing a chemo-proteomics approach. A number of putative off-target inhibitor interactions were revealed, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme Quinone Reductase 2 (QR2). Accordingly, the results described in Chapter 2 provide insight into the molecular mechanism of action of the inhibitors, as well as drug specificity, which will assist in the development of more specific next-generation CK2 inhibitors. The convergence of protein kinases and caspase signaling pathways has recently emerged, as phosphorylation of a number of caspase substrates within the caspase recognition motif has been shown to prevent caspase cleavage. Evidence of a structural mechanism for phosphorylation-dependent protection of caspase substrates, as well as the protection of a wide variety of caspase targets by multiple protein kinases, suggests a 27

global role for phosphorylation as a mechanism of regulation of apoptosis. Therefore, to investigate the global role of phosphorylation in caspase signaling, a comprehensive evaluation of proteins with an overlapping protein kinase phosphorylation and caspase recognition motifs was carried out in Chapter 3. The constitutive activity and oncogenic properties of CK2, as well as the existence of a strong similarity between the caspase degradation recognition sequence and the CK2 consensus motif for phosphorylation, signify that CK2 may be involved in the global protection of a wide variety of caspase targets. This research specifically focuses on the role of CK2, as an example, where the global phosphorylation-dependent protection of caspase targets from caspase-mediated cleavage negatively regulates caspase-signaling pathways. In Chapter 3, an investigation of the global role of phosphorylation in the regulation of caspase signaling pathways was carried out. To test this mechanism of regulation, a comprehensive evaluation of overlapping CK2 and caspase targets was performed using a sequence-based Protein Identification Targeting Strategy (sPITS). The strategy consisted of combining the global strengths of bioinformatics with the throughput capacities of peptide array target screens and the use of a novel method for identifying caspase targets, Caspase Substrate Identification (CSI). sPITS offers a powerful tool for the high throughput identification of widespread protein kinase and/or protease substrates. A number of peptides corresponding to proteins containing an overlapping CK2/caspase consensus were shown to be phosphorylated in the CK2 peptide array target screens, as well as cleaved by caspases using CSI, representing novel candidate CK2/caspase targets. Of particular interest, was the identification of an overlapping CK2/caspase motif within the caspase proteolytic activation site of pro- caspase-3. Evidence that phosphorylation at the P2 or PI' residues within the caspase cleavage recognition motif is sufficient to prevent caspase hydrolysis, as well as the identification of numerous candidate CK2/caspase targets in the present studies, support a role for phosphorylation as a global mechanism of regulation of caspase signaling pathways. In Chapter 4, validation of the phosphorylation-dependent protection of pro- caspase-3 by CK2 was carried out. In vitro studies demonstrated that pro-caspase-3 was phosphorylated by CK2 at Thrl74 and Serl76 and that the phosphorylation protected pro- 28 capase-3 from caspase-8 and -9 mediated cleavage, providing a novel mechanism for the regulation of caspase-3 activation. Identification of CK2-dependent phosphorylation of pro-caspase-3 in cells, and the discovery of a CK2/pro-caspase-3 complex, as well as the increase in apoptosis and cleavage of caspase-3 following CK2 inhibition supports a role for CK2 in caspase-3 signaling. Involvement of CK2 in the regulation of pro-caspase-3 activation, as well as in the protection of caspase-3 targets from cleavage provide a novel molecular mechanism by which CK2 protects cells from apoptosis. Chapter 5 focuses on the re-evaluation of the use of CK2 inhibitors to study CK2 function in cells and reiterates the importance of validation strategies. The combination of the use of inhibitor-resistant rescue experiments and analog-sensitive CK2 mutants is highlighted as a potential strategy to comprehensively identify cellular CK2 targets. Also noted in Chapter 5 is the potential use of inhibitor-resistant mutants and the chemical genetic approach in the validation of the numerous promising novel CK2/caspase targets identified using sPITS, including proteins involved in apoptosis and transcriptional regulation. Prospective experimental directions are outlined in Chapter 5 investigating the functional and dynamic relationship between CK2 and pro-caspase-3 in cells. The use of proteomic and mass spectrometry strategies such as SILAC, as a powerful tool to quantitate changes in pro-caspase-3 interacting partners within caspase signaling pathways during the progression of apoptosis are described. Finally, a global model of CK2 in the regulation of caspase signaling is proposed, where CK2 phosphorylation functions in the regulation of the progression of caspase cascades, via inhibiting the proteolytic activation of pro-caspases, as well as in the global protection of caspase targets from caspase-mediated cleavage.

Overall, these studies provide new insights into the molecular mechanisms by which CK2 promotes tumorigenesis and will contribute in the development of new strategies to target CK2 as anti-cancer therapeutic target. 29

1.12 References

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CHAPTER 2 AN UNBIASED EVALUATION OF CK2 INHIBITORS BY CHEMO-PROTEOMICS: CHARACTERIZATION OF INHIBITOR EFFECTS ON CK2 AND IDENTIFICATION OF NOVEL INHIBITOR TARGETS1

2.1 Introduction Targeting protein kinases for therapeutic applications has become a rapidly evolving field with a number of successful inhibitors in clinical studies or approved for treatment of various cancers. Loss of regulation of protein kinase activity has been identified as a key event in the development of cancer, and has, therefore, become an avenue of intense research. Specifically, the success of targeted therapies such as Gleevec (Imatinib) for the treatment of chronic mylogenous leukemia (CML) has provided a model for the development of novel inhibitors targeting a wide variety of protein kinases [1]. Recently, protein kinases have emerged as some of the most promising drug targets, representing as much as 20% of the 'draggable' genome [2, 3]. Ongoing pre-clinical studies targeting serine/threonine kinases have been carried out demonstrating the potential of kinase inhibitors as anti-cancer therapeutics, including the study of protein kinase CK2. CK2 (formally known as casein kinase II), is a ubiquitously distributed, constitutively active serine/threonine kinase implicated in multiple cellular processes, including cell survival, protection of cells from apoptosis and tumorigenesis [4]. Elevated CK2 activity has been documented in a number of cancers, where high CK2 activity has been correlated with aggressive tumor behavior [5]. Detailed studies have been carried out to elucidate the molecular mechanism of CK2 in tumorigenesis, demonstrating that increased CK2 activity promotes a number of pathways participating in the development of cancer [6, 7]. Proposed modes of action of CK2 in tumorigenesis are through the regulation of key oncongenes and tumor suppressor proteins within various pro-survival

pathways including the Wnt [8], NF-KB [9], and PI3-K pathways [10]. Regulation of

1 This chapter has been published: Duncan JS, Gyenis L, Lenehan J, Bretner M, Graves LM, Haystead TA, Litchfield, DW. An unbiased evaluation of CK2 inhibitors by chemo-proteomics: Characterization of inhibitor effects on CK2 and identification of novel inhibitor targets. Mol Cell Proteomics. 2008 June; 7(6): 1077-88 44 oncogene and tumor suppressor proteins by CK2 phosphorylation has been shown to influence susceptibility to proteosome degradation, protect proteins from caspase- mediated cleavage and alter the protein activity [11]. Taken together, elevated CK2 activity promotes a pro-survival signal in cancer by facilitating the transcription, activation or stability of oncogenes and anti-apoptotic proteins, while repressing tumor suppressor activity through increased proteosome degradation. Recently, an anti- apoptotic role of CK2 has emerged as a number of substrates were identified that exhibited protection from caspase cleavage following CK2 phosphorylation [12-16]. Consequently, CK2 has emerged as a promising target for therapeutic intervention in the treatment of cancer. Development of specific ATP-competitive protein kinase inhibitors has been historically difficult, as many cellular proteins utilize ATP as a substrate; however, exploitation of unique structural aspects of kinases has provided an avenue to develop highly selective inhibitors. A number of ATP-competitive inhibitors have been developed to target CK2 based on the solved crystal structure, which shows unique structural properties that distinguished CK2 from the majority of other kinases [17]. Most notably, CK2 was found to have a relatively small ATP binding pocket, compared with other protein kinases, due to the presence of large bulky residues that are essential for ATP binding. Exploitation of these distinctive bulky residues within the ATP-binding pocket has provided opportunities to develop highly-selective CK2 inhibitors. Based on the structure of a known CK2 inhibitor DRB (5,6-Dichlorobenzimidazole ribofuranoside), a novel compound TBB (4,5,6,7-Tetrabromo-lH-benzotriazole) was engineered [18], which displayed a high affinity for CK2 due to strong hydrophobic and Van der Waals interactions with the large bulky residues in the ATP binding pocket (Figure 2-1). TBB exhibited high selectivity for CK2 in a panel of 30 kinases, however, there were a handful of kinases that were sensitive to the inhibitors, including DYRKla, which was inhibited to nearly the same extent as CK2, while GSKp\ CDK2 and PHK were inhibited to a lesser extent. Therefore, strategies to improve the selectivity of the inhibitors were undertaken focusing on generating compounds that had improved interaction with the unique bulky residues. Utilizing the TBB backbone as a scaffold, two next generation derivatives were engineered, TBBz (4,5,6,7-tetrabromo-lH-benzimidazole) and DMAT (2- 45

Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole) [19, 20]. In vitro studies testing the efficacy of DMAT showed that substitution of the imidazole ring and the addition of

MM.

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Figure 2-1. Chemical structure of ATP and commercially-available CK2 inhibitors. IC50 and Kj values in uM concentrations. 46 bulky constituents resulted in a higher affinity and selectivity towards CK2 with a K; value of 40 nM. In this study, an unbiased evaluation of TBB and related CK2 inhibitors utilizing chemo-proteomics was carried out to investigate the potential of the inhibitors as anti cancer therapeutics. A detailed study of the specificity of the CK2 inhibitors TBB, TBBz and DMAT for CK2, as well as the cellular effects associated with each inhibitor was carried out. Differences between inhibitors in the induction of apoptosis was observed following treatment of HeLa cells with TBB, TBBz or DMAT, indicating that the inhibitors have unique biological properties, as well as revealed the potential for off- target effects. Therefore, rescue experiments utilizing inhibitor-resistant CK2 mutants were employed to investigate the specificity of the inhibitors, particularly, whether the apoptosis associated with drug treatment was CK2 dependent. The inability to rescue TBBz- and DMAT-induced apoptosis with the resistant mutants prompted a systematic characterization of inhibitor protein targets utilizing a chemo-proteomics approach.

2.2 Materials and Methods 2.2.1 Cell culture and Transfections HeLa and U20S cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco), penicillin (100 U/mL) and streptomyocin (100 [xg/mL) (Gibco) on 10 or 15 cm plates (Falcon). HeLa and U20S cells were transfected with either wild type CK2a/a', inhibitor-resistant CK2aR/a'R, or kinase inactive CK2cc'-KD, in addition to CK2|3 / CK2p6KR using calcium phosphate with a transfection efficiency > 80%. Cells were treated with TBB (Calbiochem), TBBz (Sigma) or DMAT (Calbiochem) at a final concentration of 8-25 uM for 6, 12, 18 and 24 hrs. Microscopic images were captured on an inverted microscope (Zeiss, Axiovert S25) using a mounted digital camera (QiCAM Q imaging). HeLa S3 suspension cells were cultured in MEM (Sigma) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 [xg/mL streptomyocin. Cells were grown to confluency and harvested for use in chemo-proteomic experiments. 47

2.2.2 Cloning of CK2aR and CK2a 'R Inhibitor-resistant CK2 mutants were engineered using a QuickChange II Site- Directed Mutagenesis Kit (Stratagene). Human CK2o>HA pRc/CMV was PCR-amplified to make the V66A mutation using the primer 5'-GAAAAAGTTGCAGTTAAAATTC-3' and the I174A mutation using 5'-GCTACGACTAGCAGACTGGGGTTTGGC-3'. Human HA-CK2cc' pRc/CMV was PCR-amplified to make the V67A mutation using the primer 5'-ATGAGAGAGTGGCTGTAAAAATCCTGA-3' and I175A mutation using 5 '-AAGCTGCGACTGGCAGATTGGGGTCTG-3'. 2.2.3 Flow Cytometry HeLa cells were trypsinized at 18 hrs following treatment with 8 or 25 |oM CK2 inhibitors. Cells were washed in ice-cold PBS and filtered using a cell strainer (VWR) to obtain single-cell suspensions. Cells were fixed by the addition of ice-cold 95% ethanol and stored at -20°C. Cells were then washed in PBS and the DNA was stained with PI staining solution containing 50 ug/ml propidium iodide (Sigma), 0.1% sodium citrate, 0.1% Triton X-100, and 100 [xg/ml RNase A in the dark for 1 hr at 37 °C. Cells were diluted with PBS to 1 mL and analyzed by flow cytometry (FACSCaliburs). Spectra and statistics representing the amount of DNA in different cell cycle stages were prepared using FlowJo Flow cytometry analysis software. 2.2.4 Western Analysis HeLa and U20S cells were harvested from 10 and 15 cm plates (Falcon) in lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.0%

Triton-X-100, 0.5% NP40, 2.5 mM sodium pyrophosphate, 1 mM Na3V04, 1 mg/ml Leupeptin and 1 mM PMSF. Lysates were sonicated and protein concentrations were determined by BCA assay. Proteins were resolved on 12% SDS-PAGE electrophoresis followed by transfer to PVDF membranes (Roche). Membranes were blocked with 5% BSA/PBST, 5% BSA/TBST or 5% nonfat milk/TBST for 1 hrs, followed by an overnight incubation at 4 °C with the primary antibody in 5% BSA TBST or 5% nonfat milk/TBST. Membranes were probed with primary antibodies including: anti-PARP 1/1000 (Cell Signaling Technologies), anti-HA 3F10 1:100 (Roche), anti-cMyc 9E10 1:10000 (Berkeley Antibody Company), |3-tubulin 1:100 (Sigma), HSP90 1:5000 (Santa Cruz Biotechnology), anti-CK2a 1:5000 polyclonal antiserum directed against the C-terminal 48 synthetic peptide a and anti-CK2a' 1:5000 polyclonal antiserum directed against the C-terminal synthetic peptide a' " . Membranes were washed with PBST or TBST then incubated in appropriate secondary antibodies, including: (BioRAD) HRP-GAR (1:2000 for PARP, 1:25000 for HSP90 and CK2a and CK2cx' antibodies), HRP-GAM (BioRAD) (1:1000 for (3-tubulin) and HRP-Biotin (Jackson ImmunoResearch) (1:10000 for HA 3F10 and cMyc 9E10). Following secondary antibody incubation, membranes were washed with PBST or TBST and visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia). X-ray film (Kodak) was developed and converted to a digital image using CanoScan N650U/N656U scanner. Images were visualized in Adobe Photoshop CS. 2.2.5 Immunoprecipitations HeLa cells were co-transfected with wild type CK2o>HA or inhibitor-resistant CK2ctR-HA, myc-CK2|3 and EGFP-C2 using calcium phosphate with a transfection efficiency > 80%. Following transfection, cells were treated with DMSO, 8 uM TBB, TBBz or DM AT for 18 hrs. To test the ability of CK2a'R to rescue inhibitor dependent changes, U20S cells were co-transfected with HA-CK2a'-wt or HA-CK2aR and myc- CK2(3, then treated with 25 uM DMSO or DMAT for 12 hrs. Cell lysates were prepared in RIPA lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 1% NP40, 0.5% Deoxycholic acid, 0.1% SDS and 1 mM EDTA) and the protein concentration was determined by BCA assay (Pierce). Equal amounts of protein were incubated with 20 uL of 50% protein A- Sepharose beads (Amersham Pharmacia) and 1-5 \ig 12CA5 HA antibody (Roche). Following a 1 hr incubation tumbling at 4°C, protein A-sepharose beads were isolated by centrifugation and washed extensively. Beads were then incubated in SDS loading buffer, boiled for 5 min, and centrifuged briefly to remove beads. Proteins were resolved on a 12% SDS PAGE gel, transferred to a PVDF membrane, followed by Western blot analysis as described previously. Membranes were probed with biotinylated anti-HA 3F10 and anti-cMyc 9E10 antibodies. 2.2.6 ATP-Sepharose affinity chromatography HeLa S3 cells cultured in 1 L spinner flasks were lysed in buffer A (150 mM

NaCl, 50 mM HEPES, 10 mM MgCl2, 1% NP40, 1 u.g/mL leupeptin, 1 ug/mL aprotinin, 1 mM dithiothreitol) and incubated on ice for 10 min. Lysates were sonicated and 49 subjected to centrifugation at 80,000 g for 30 min at 4 °C. Equal volumes of lysates were incubated with DMSO and either 1 uM TBBz or 100 uM TBB, TBBz or DMAT for 2 hrs with gently rocking at 4 °C. ATP-Sepharose beads (as described earlier, [21]) were then added to the lysates containing the inhibitors and incubated at 4 °C for 15 min to allow for binding. ATP-Sepharose beads were isolated by centrifugation and washed extensively with 100 column volumes of buffer A, followed by a wash with 1 M NaCl buffer A and finally beads were equilibrated with buffer A. Equilibrated ATP-Sepharose beads were then incubated in 2D lysis buffer for 15 min with gentle rocking at 4 °C. Protein concentrations were determined by Bradford assay. Equal concentrations of protein from each treatment were run on ID and 2D gel electrophoresis, as described previously. 2.2.7 2D gel electrophoresis Following cell synchronization and inhibitor treatment as described previously, cells were harvested from 15 cm plates using PBS-EDTA and collected by centrifugation. Proteins were purified via TRIzol extraction according to Lysis and Protein Extraction from 32P-Labeled WEHI Cells with TRIzol Isolation Reagent, AfCS Procedure Protocol ID PP00000155. Purified proteins were re-suspended in 2D lysis buffer containing 7 M Urea, 2 M Thiourea, 4% (w/v) CHAPS, 1 mM Benzamidine, 2.5 mg/mL, 20 u.g/mL

Pepstatin, 10 u.g/mL Aprotinin, 1 mM Dithiothreitol (DTT), 200 mM Na3V04, 100 yM Microcystin, 0.5% (v/v) Ampholines pi 4-7 buffer (Amersham Pharmacia). Protein concentrations were determined by Bradford assay (Bio-Rad). Protein samples (150 \x,g- 250 |Ag) were mixed with rehydration buffer containing 7 M Urea, 2 M Thiourea, 4% CHAPS, 50 mM DTT, 0.5% Ampholines and bromophenol blue then applied to Immobiline Dry strips (Amersham Pharmacia, 7 cm pi 4-7 or 13 cm pi 3-10) in ceramic strip holders (Amersham Pharmacia). The first dimension isoelectric focusing was carried out using the IPGphor II (Amersham Pharmacia) using the following program for the 7cm IPG strips; rehydration step 1, 20 V 12 hrs at 20 °C, step 2, 100 V for 100 V-h, step 3, 500 V for 500 V-h, step 4,1000 V for 1000 V-h, step, 5 2000 V for 2000 V-h, step 6, 4000 V for 4000 V-h, step 7, 8000 V for 16000 V-h and for the 13 cm strip; rehydration step 1, 20 V 12 hrs at 20°C, step 2,100 V for 2 hrs, step 3, 500 V for 500 V- h, step 4, 1000 V for 1000 V-h, step 5, 2000 V for 4000 V-h, step 6, 4000 V for 8000 V- h, step 7, 6000 V 12000 V-h, step 8, 8000V for 64000 V-h. The IPG strips were then 50 incubated in equilibration buffer containing 10 nig/mL DTT (Sigma) followed by 25 mg/mL iodoacetamide (Sigma). The 7 cm IPG strips were then applied to a 12% SDS- PAGE gel, sealed with 0.5% agarose gel and run at 200 V for 1 hr (Bio-Rad Mini-Protean 3 Dodeca Cell), while the 13 cm IPG strips were run on 10% SDS-PAGE gels (HOFFER scientific instruments). Following protein separation, gels were fixed and stained with SYPRO-RUBY (Invitrogen) overnight at room temperature. Stained gels were washed and imaged using the ProXPRESS 2D Proteomic imaging system (Perkin Elmer). 2.2.8 2D gel electrophoresis quantification and analysis Differences in protein spots between 2D gels were analyzed utilizing Phoretix 2D evolution software (TT900 S2S, PG220 Nonlinear Dynamics). Four gel images from each treatment were imported into Phoretix software and spots were aligned using SameSpot TT900 S2S same spot analysis. The aligned images were then imported into Progenesis PG220 software for analysis of spot differences and spot intensity. Spot filtering was applied to all aligned gels to normalize for pixel intensity and area with a normalized volume value of > 0.005. Statistical analysis of spot changes were performed using ANOVA and the means were compared with a T-test where p = 0.05. 2.2.9 Sample preparation and mass spectrometry Following staining of 2D gels with SYPRO RUBY and identification of spot differences using Phoretix evolution; spots were picked from gels manually or by the Ettan Spot Picker (Amersham) and suspended in 50% methanol and 5% acetic acid. Trypsin digestion was performed on excised spots using the MassPREP automated digestor (Waters). Peptides were lyophilized, suspended in 30% ACN/0.1% TFA mixed with cc-cyano-4-hydroxycinnamic acid (CHCA) in 50%> ACN/50% 25 mM ammonium citrate/0.1% TFA and analyzed by MALDI MS and/or MS/MS on the 4700 proteomics analysis MALDI (TOF:TOF) instrument (Applied Biosystems). MS and/or MS/MS analysis was carried out with an m/z range of 800-4000 Da and mass tolerance of 50 ppm with a resolution of approximately 15 000. Peptide fingerprinting was evaluated using GPS Explore Workstation version 3.0 series (Applied Biosystems) in conjunction with MASCOT using an internal calibration with a min S/N threshold of 20, peptide mass exclusion tolerance of 50 ppm, 5 minimum peaks to match and a maximum outlier error of 15 ppm with no more than 1 missed cleavage. Mass references consisted of des-Argl- 51

Bradykinin (904.468), Angiotensin 1 (1296.685), Glul-Fibrinopeptid (1570.677), ACTH (1-17) (2093.087), ACTH (18-39) (2465.199), while the mass exclusion list consisted of (842.5099, 870.509, and 2211.1096). Peptides were searched against the human Swiss-Prot database (release 54.0 July 24 2007, 276,256 sequence entries) with oxidation as a variable modification (M).

2.3 Results 2.3.1 Effect of CK2 inhibitors on cell viability: Evidence for unique modes of drug action Treatment ofHeLa cells with CK2 inhibitors induces apoptosis Pharmacological inhibition of CK2 has been shown to sensitize various cancer cell lines to apoptosis, as well as induce apoptosis directly [19, 22-24]. A number of highly-specific chemical compounds have been developed to target CK2 in cells including TBB and its derivatives, TBBz and DMAT. To further characterize the effect of these TBB-derived inhibitors on cancer cell viability, an evaluation of inhibitor- induced apoptosis was performed utilizing HeLa cells (Figure 2-2A). The cleavage of Poly(ADP-ribose) polymerase (PARP) was used as a biological marker of apoptosis and caspase activation. Etoposide, a known apoptotic stimulus, was used as a positive control, while DMSO, the drug carrier for all the inhibitors being investigated was used as a negative control. Induction of apoptosis was evident by the production of cleaved PARP following 24 hrs of treatment with 25 \xM etoposide, TBB, TBBz and DMAT. Interestingly, the induction of apoptosis was observed following 12 hrs of treatment with TBBz and DMAT but not until 24 hrs following treatment with the same concentration of TBB. It appears that the different derivatives have varying abilities to induce apoptosis at the same concentration, where TBBz and DMAT were the most potent inhibitors. To further characterize the biological responses of cells treated with CK2 inhibitors, TBB, TBBz and DMAT, we examined changes in protein profiles using 2D gel electrophoresis. HeLa cells were treated with 25 uM TBB, DMAT, TBBz or DMSO for 12 hrs, subjected to 2D gel electrophoresis, and the resultant spot changes were analyzed using 2D Phoretix software. Spot differences between treatments were identified (spots 1- 4), excised from the gels and subjected to mass spectrometry for protein identification. 52

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Figure 2-2. CK2 inhibitor-dependent induction of caspase-mediated apoptosis. (A) HeLa cells treated with DMSO and either 25 uM etoposide, TBB, DMAT or TBBz for 6, 12 and 24 hrs were harvested and the protein lysates were resolved by 12% SDS PAGE. Proteins were transferred to PVDF membranes and analyzed via Western blot probing for PARP (116 kDa) and |3-tubulin (55 kDa). Cleaved PARP is indicated on the blots as an 89 kDa band which is an indicator of apoptosis (B) HeLa cells were treated with DMSO or 25 uM TBB, DMAT or TBBz for 12 hrs; proteins were then purified by TRIzol extraction and run on 7 cm pi 4-7 IPG strips for the first dimension of isoelectric focusing. Focused proteins were then run on 12% SDS PAGE gels for the second dimension, followed by SYPRO RUBY protein staining. Sections from representative 2D gels were shown to illustrate changes in spot patterns between treatments. Spots 1, 2 and 3 were identified by MALDI MS as Vimentin (MW 53.7 kDa, pi 5.06) caspase cleavage products, while spot 4 was identified as a Keratin K18 (MW 48.06 kDa, pi 5.34) caspase cleavage product. 53

Spot 1 (~ 48 kDa), spot 2 (~ 45 kDa) and spot 3 (~ 25 kDa) were identified in the TBBz and DMAT (but not TBB or DMSO gels) as Vimentin proteolytic products by MS analysis (Figure 2-2B). The fragments generated were consistent with previous findings demonstrating a caspase-dependent apoptosis, where Vimentin (57 kDa) was found to be cleaved by caspases 3, 6 and 7 into fragments with molecular masses of 48, 45 and 25 kD [25]. Keratin Kl 8 was also identified as a proteolytic fragment (spot 4, ~22 kDa) present in the TBBz-and DMAT-treated gels (but not TBB or DMSO) by MS analysis. The cleavage of Keratin 18 (48 kDa) by caspases 3, 6 and 7 during apoptosis has been shown in previous studies, resulting in the generation of proteolytic fragments, including a 22 kDa peptide [26]. Overall, an analysis of the proteome of TBBz- and DMAT-treated HeLa cells provided further evidence for inhibitor-dependent apoptosis, as well as suggested that the apoptosis stimulated by TBBz and DMAT were mechanistically similar requiring caspase activation. The induction of apoptosis by TBBz and DMAT was further investigated revealing a dose-dependent effect, where increasing drug concentration resulted in a more potent apoptotic response, as is evident by an amplified cleavage of PARP (Figure 2-3). The potent induction of apoptosis following TBBz and DMAT treatment suggests a unique mechanism of action relative to TBB.

Figure 2-3. Concentration- and duration-dependent induction of apoptosis following CK2 inhibitor treatment. HeLa cells were treated with DMSO and 1, 5, 10, 15 and 25 \xM TBBz or DMAT for 18 hrs. Proteins were transferred to PVDF membranes and analyzed via Western blot probing for PARP. 54

2.3.2 Employing inhibitor-resistant CK2 mutants to test drug specificity Validation ofCK2 as an inhibitor target in cells The potency of a number of CK2 inhibitors to induce apoptosis in various cancer cells raises promising avenues for targeting CK2 for anti-cancer therapeutics. Studies performed in vitro utilizing CK2 inhibitors have demonstrated their high effectiveness at inhibiting CK2 kinase activity. However, the specificity and mechanism of action of these ATP-competitive inhibitors has not been systematically explored in an unbiased manner. To investigate the ability of TBB, TBBz and DM AT to inhibit CK2 activity in cells, the autophosphorylation of CK2|3 by CK2a/a' was examined. The autophosphorylation of CK2(3 has been shown to be essential for its stability, and a marker of assembly of CK2 tetrameric complexes. Therefore, autophosphorylated CK2(3 exists predominantly in complex with CK2a/a' in the phosphorylated form [27-30]. In vitro studies, testing the effectiveness of CK2 inhibitor-resistant mutants have been performed and demonstrated that the mutants are fully functional and resistant to TBB, TBBzandDMAT[17,24]. To extend the analysis of the inhibitors and to validate drug specificity, HeLa cells co-expressing wild type CK2a-HA or resistant CK2aR-HA in addition to Myc- CK2(36KR (Mycp) were treated with either 8 \M TBBz, DMAT or TBB, as well as a DMSO control, for 18 hrs (Figure 2-4). The proteasome-resistant Myc-CK2|36KR mutant was used to investigate CK2 tetrameric complex formation [31]. Wild-type CK2a and resistant CK2aR were immunoprecipitated from treated cells and the levels of complexed Myc(3-P; were analyzed. Consistent with previous findings, treatment of cells expressing wild type CK2 in the presence of the CK2 inhibitors resulted in the reduction of CK2a- MycP-Pi complex formation, as compared to treatment with the drug carrier DMSO, as was evident by the reduced CK2a and Myc|3-Pj levels. Interestingly, the expression of the inhibitor-resistant CK2 rescued the loss of CK2a/Myc|3-Pi complex formation as was evident by stable formation of CK2a/Myc|3-Pi complexes and increased CK2aand Myc|3-Pj levels. Overall, introduction of the inhibitor-resistant CK2aR rescued the reduction of CK2a and Myc|3-Pj levels in the tetrameric complex, indicating that 55

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Figure 2-4. Employing inhibitor-resistant CK2a mutants to test drug specificity. (A) Rescue of CK2|3 autophosphorylation following treatment with CK2 inhibitors using inhibitor-resistant CK2 mutants was demonstrated in HeLa cells co-transfected with CK2o>HA or CK2ccR-HA, in addition to myc-CK2p6KR (Myc|3) and EGFP-C2 at a transfection efficiency > 80%. Cells were treated with DMSO or 8 \xM TBBz, DMAT or TBB for 18 hrs. CK2a-HA and CK2aR-HA were immunoprecipitated from lysates, run on a 12% SDS PAGE gel followed by transfer to a PVDF membrane. A decrease in the formation of tetrameric complexes, as well as complexed Mycp-Pj in the presence of TBBz, DMAT and TBB was indicated by the reduction of the 32.5 kDa Mycp-P; band and the 50 kDa CK2a-HA band as shown via Western blot analysis by probing membranes with anti-Myc 9E10 and anti-HA 3F10 antibodies, compared to the control DMSO. The rescue of the formation of CK2 tetrameric complexes and loss of mycp-P; in the presence of the inhibitors was demonstrated by restoring the 50 kDa CK2a-HA and the 32.5 kDa MycP-Pj bands back to DMSO levels. The levels of Myc|3-P; in cells prior to immunopreciptations were shown by Western blot analysis as a control. 56

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Figure 2-5. Employing inhibitor-resistant CK2a' mutants to test drug specificity. To functionally test the inhibitor-resistant CK2a'R mutants, U2-OS cells expressing wild- type CK2a', or resistant CK2ct'R in addition to myc-CK2|3 were treated with DMSO or 25 \iM DMAT for 12 hrs. Cells expressing kinase-inactive CK2a'-KD were used as a control for loss of CK2 activity. A decrease in the formation of tetrameric complexes, as well as complexed Myc|3-Pj in the presence of DMAT was indicated by the reduction of the 32.5 kDa Myc|3-Pi and 43 kDa HA-CK2a' bands as shown via Western blot analysis by probing membranes with anti-Myc 9E10 and anti-HA 3F10 antibodies, compared to the control DMSO. The inhibitor-resistant CK2a'R mutant rescued the formation of the tetrameric complexes and loss of CK2(3 autophosphorylation in the presence of DMAT which was observed by the restoration of the 43 kDa HA-CK2a'R and the 32.5 kDa Mycp-Pj bands back to DMSO control levels. Restored levels of HA-CK2a'R and Myc(3- Pi were detected by Western blot analysis probing membranes with anti-Myc 9E10 and anti-HA 3F10 antibodies. In accordance, a decrease in tetrameric complex formation and complexed Myc|3-Pj levels were observed with the kinase-inactive CK2a'-KD mutant. 57 restoration of CK2a kinase activity was capable of autophosphorylating CK2|3, thus stabilizing CK2a/CK2|3 complex formation. Inhibitor-resistant CK2a' (V67A/I175A) mutants were also shown to be capable of forming functional tetrameric complexes, as well as being proficient at restoring the loss of autophosphorylation of Myc(3 associated with inhibitor treatment (Figure 2-5). Overall, the treatment of cells with CK2 inhibitors TBB, TBBz and DMAT resulted in the inhibition of autophosphorylation of CK2|3 by CK2a/cc'. The rescue of the autophosphorylation of CK2|3 by CK2aR/a'R validates that the inhibitors reduce CK2 kinase activity in cells, which can be restored by expression of resistant CK2.

Inhibitor-resistant mutants are unable to rescue apoptosis Inhibition of CK2 using a number of inhibitors including TBB, TBBz and DMAT have all been shown to negatively affect the viability of various cancer cell lines [19, 24, 32]. This was also observed in the present study, where treatment of HeLa cells with TBBz, DMAT and to a lesser extent, TBB, induced apoptosis in a concentration- and duration-dependent manner. In rescue experiments, it was shown that TBB, TBBz and DMAT inhibited the autophosphorylation of CK2(3 in cells that could be restored by expressing inhibitor-resistant CK2 mutants. To test whether the apoptotic response associated with TBB, DMAT and TBBz was due to inhibition of CK2 activity, rescue experiments utilizing inhibitor resistant CK2 were carried out. HeLa cells expressing CK2aR and CK2a'R were treated with DMSO, 8 [xM TBB, DMAT or TBBz for 18 hrs, and apoptosis was determined by assaying for cleaved PARP by western blot (Figure 2-6A) and by the determination of the percentage of cells in the sub-Go/Gi population via FACS (Figure 2-6B). Interestingly, cells expressing the inhibitor resistant forms of CK2 still showed evidence of apoptosis following treatment with TBBz or DMAT, indicated by the presence of cleaved PARP, as well as a large percentage of cells in the sub-Go/Gi population, compared to DMSO controls. By comparison no apoptosis was observed in TBB-treated cells either with or without expression of resistant CK2. Overall, this evidence suggests that the apoptosis associated with TBBz and DMAT was not exclusively due to loss of CK2 activity, as restoration via expression of inhibitor-resistant mutants was not capable of rescuing cells 58

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1 DMAT BD9- |l DMAT + TBB !] TBB + CK2 R ll CK2-R '% s ,i !-..«,- I sM' '--"--.,_J .fill — i

Figure 2-6. Apoptosis associated with TBBz and DMAT treatment was independent of CK2 activity. (A) TBBz- and DMAT-induced apoptosis could not be rescued by restoring CK2 activity in cells. HeLa cells were co-transfected with CK2aR-HA/HA-CK2a'R, along with myc-CK2(36KR and EGFP-C2 at a transfection efficiency > 80%, and treated with either DMSO or 8 uM TBBz, DMAT or TBB for 18 hrs. Apoptosis was determined by the presence of the 89 kDa cleaved PARP band in TBBz- and DMAT-treated cells expressing inhibitor-resistant CK2 mutants, compared to DMSO. No cleavage of PARP was detected in cells treated with TBB. Confirmation of expression of resistant mutants was determined by the presence of a 50 kDa CK2aR-HA and 40 kDa HA-CK2a'R bands. Western blot analysis was performed using anti-PARP, anti-HA 3F10 and |3- tubulin antibodies. (B) Inhibitor induced apoptosis in HeLa cells co-transfected with CK2ctR-HA/HA-CK2a'R, along with myc-CK2p6KR was assayed by FACS via the determination of the percentage of cells in the sub-Go/Gi population. FACS samples were prepared as described earlier. A reduction in the percentage of cells in the sub- Go/Gi population of apoptosis was not observed in cells expressing resistant CK2 following treatment with 8 \iM TBB, DMAT or TBBz. 59 from apoptosis. Interestingly, treatment of cells with 8 uM TBB resulted in a reduction of CK2 activity, as was evident by the loss of CK2|3 autophosporylation but no induction of apoptosis. The potency of TBBz and DMAT in inducing a CK2-independent apoptosis, even at low drug concentrations, raises the possibility of off-target drug effects and cytotoxicity. In this respect, evidence presented here suggests that the death associated with TBBz and DMAT may not be attributed solely to the inhibition of CK2 but rather the inhibition of other ATP-binding proteins that are essential for cell viability.

2.3.3 Identification of novel inhibitor interactors: Evidence for off-target effects Confirmation ofCK2 as a bona fide target of TBB and its derivatives To validate CK2 as an inhibitor target, a chemo-proteomic strategy was employed. Accordingly, a proteomic approach employing ATP-Sepharose affinity chromatography was performed to test the specificity of TBB, TBBz and DMAT for CK2. An inhibitor competition assay was performed, consisting of pre-incubation of HeLa S3 cell lysates with DMSO, 100 jxM TBB, DMAT or TBBz, followed by the addition of ATP-Sepharose beads. If the inhibitor interacted with an ATP-binding protein with strong affinity it would prevent that protein from binding to the ATP-Sepharose. To test whether CK2 was a bona fide target of TBB, DMAT or TBBz amongst all cellular ATP-binding proteins, a comparative analysis of the proteins bound to the ATP-Sepharose beads following inhibitor treatment was performed via Western blot analysis for CK2a and CK2cc' (Figure 2-7A). Incubation of HeLa cell lysates with 100 \xM TBB, DMAT or TBBz prevented CK2a and CK2a' from binding to the ATP-Sepharose, relative to the control DMSO levels. Interestingly, the reduction in CK2ct' immuno-reactivity observed in the presence of 100 uM TBBz could be restored to control levels by reducing the inhibitor concentration to 1 uM, indicating a concentration dependent competition. (Figure 2-8). HSP90 was used as a loading control, which was unaffected by the inhibitor competition assay. Taken together, CK2a and CK2a' were found to interact with TBB, DMAT and TBBz amongst all other ATP-binding proteins, further validating CK2 as a bona fide target of TBB and its derivatives. 60

CK2a

CK2a'

HSP90

B DMSO TBBz TBB DMAT r 4.. jr. . i 1 2a

66 if ".-***'-

Figure 2-7. Exploiting an unbiased chemo-proteomic approach to identify CK2 inhibitor interactions. (A) HeLa S3 cells were harvested in buffer A (see experimental procedures) and incubated with DMSO or 100 uM TBBz, DMAT or TBB. An inhibitor competition assay was then performed as described in (A), followed by Western blot analysis by probing blots with anti-CK2a', anti-CK2a and anti-HSP90 antibodies. (B) A number of spot changes were observed upon CK2 inhibitor treatment, indicating a number of putative CK2-inhibitor interactions. HeLa S3 cells were harvested in buffer A (see Materials and Methods) and incubated with 100 uM DMSO, TBBz, DMAT or TBB. An inhibitor competition assay was then performed by the addition of ATP-sepharose beads to pre-inhibitor treated lysates. Following extensive bead washes, proteins were isolated and analyzed by 2D gel electrophoresis. Spot differences were determined using Phoretix 2D evolution software. Enlarged sections from representative 2D gels from DMSO, TBB, TBBz and DMAT treated lysates illustrate differences in spot patterns. The presence of protein spots on the DMSO gels and not on the inhibitor gels were compared and putative inhibitor-interactions were identified. Proteins identified as corresponding to inhibitor-dependent spot changes are listed in Table 2-1. 61

Table 2-1. Identification of putative off-target CK2 inhibitor interactions. The presence of protein spots on the DMSO gels and not on the inhibitor gels were compared and putative inhibitor-interactions were identified. Proteins identified as corresponding to inhibitor-dependent spot changes are listed. Proteins identified by MALDI MS or MALDI MS/MS. Spots 1- 3 identified on 2D gels depicted in Figure 2-7B.

Inhibitor Observed Spot Accession Observed Protein No. interactor number Mass (kDa) pi

Caseinolytic TBB Peptidase B TBBz Q9H078 79 9.0 (HSP78)a DMAT

Multifunctional TBB 2 protein TBBz P22234 52 8.8 (ADE2)fl DMAT

Quinone Reductase TBBz 2 P16083 30 6.0 DMAT (QR2)* a Protein identification by MALDI-TOF mass spectrometry * Protein identification by MALDI-TOF/TOF mass spectrometry 62

CK2a' —*

HSP90 —• ^•^|l

Figure 2-8. Concentration-dependent inhibitor interaction with CK2. (A) Inhibitors interact with CK2a' in a concentration-dependent manner. HeLa S3 cells were harvested in buffer A and incubated with DMSO, 100 uM TBBz, or 1 |J,M TBBZ. An inhibitor competition assay was then carried out by the addition of ATP-Sepharose beads to pre- inhibitor-treated lysates. Following extensive bead washes, proteins were isolated and analyzed by 2D gel electrophoresis. 2D gels were transferred to PVDF membranes and probed with anti-CK2a' and HSP90 antibodies. The reduction in the 38 kDa CK2a' band in lysates incubated with 100 \xM TBBz indicated a competition between TBBz and ATP binding, compared to the DMSO control. The restoration of the 38 kDa CK2a' band in 1 \xM TBBz-treated lysates indicated that no competition occurred, suggesting that CK2a' does not interact with TBBz at low concentrations. HSP90 (90 kDa) was used as a loading control. 63

Identification of novel TBBz and DMAT off-targets Following the validation of CK2 as a target of the TBB-related compounds, a more global analysis of inhibitor protein interactors was explored utilizing the chemo- proteomic approach. The inhibitor competition assay, as described previously was employed in combination with 2D gel electrophoresis and mass spectrometry to identify potential drug off-targets. A comparison of the proteins bound to the ATP-Sepharose beads was performed utilizing 2D gel electrophoresis and the protein patterns were analyzed using 2D Phoretix software. A number of putative inhibitor drug targets were identified by mass spectrometry as being present on the DMSO control gels and absent on the CK2 inhibitor-treated gels, (spots 1-3) (Figure 2-7B), including Caseinolytic Peptidase B (HSP78), Multifunctional protein ADE2, and the detoxifying protein, Quinone Reductase 2 (QR2) (Table 2-1). QR2 (spot 3), was identified as being present in the DMSO- and TBB-treated gels but not in the TBBz- and DMAT-treated gels (Figure 2-9A). This suggests that an inhibitor competition occurred, providing evidence for a novel TBBz and DMAT drug target. The spot intensity values were analyzed by ANOVA to determine statistical significant differences (p = 0.003) and treatment means were compared using a T-test (Figure 2-9B). Spot #3 (QR2) from the DMSO and TBB gels was subjected to MALDI MS analysis, which produced a prominent peak with a mass of 1986.10 (Figure 2-9C). MALDI MS/MS was carried out on the 1986.10 Da peptide to obtain protein sequence data (Figure 2-9D). The trypsin peptide fragments were analyzed by Mascot and Quinone Reductase 2 (QR2) (MW 25.6 kDa, pi 5.88) was identified with a sequence coverage of 46% (Figure 2-9E). The use of a chemo- proteomic strategy to identify TBB, TBBz and DMAT protein targets revealed that CK2 is a target for the inhibitors; however, discovery of novel metabolic targets that interact strongly with TBBz and DMAT raises the issue of the specificity of these CK2 inhibitors.

2.4 Discussion The role of CK2 in the progression of tumorigenesis has become increasingly evident, as the dysregulation of a number pathways have been attributed to abnormally high levels of CK2 activity. Consequently, pre-clinical studies investigating CK2 as a molecular therapeutic target have been ongoing, resulting in the development of various 64

B DMAT DMSO p*-|fJU 1p < 0.005

I •*• ' *• !£••£ 1 1 DMAT DMSO TBB TBBz TBB TBBz D

1*86.10 VLAPQISFAPEIASEER

304.17

175.12 v 833« 1058.55 V 433.21 «,/ «,/

E Peptide match summary for Quinone Reductase 2 (QR2)

1 MM3KKVLIVY AHQEPKSFN8 SLKNVAVDEL SRQOCTVTVS DLYAMHLEPR SI ATDKDITQTL SNPEVFNYG7 ETHEAYKQRS IASDITDEQK K7RIADLVIF 101 QFPLYUFSVP AILKOHHDHV LCQSFAfDIP OFTDSSIiLQO KLALLSVTTG 151 GTAEMYTCTO VHGMRWHI PLQBOTLHFC 0FKVLAPQIS FAPEIASEEE 201 KKSMVAAWSQ RLQTimCEEP IPCTAHHHFG Q

Figure 2-9. Identification of a novel TBBz and DMAT off-target, Quinone Reductase 2. (A) Enlarged representation of spot 3 (QR2) identified from the 2D gels shown in Figure 5B. The absence of QR2 from TBBz- and DMAT-treated gels indicates that the CK2 inhibitors prevented QR2 from binding to ATP-Sepharose, indicating an inhibitor QR2-interaction. (B) A statistical difference in QR2 binding to ATP-Sepharose between DMSO, TBB, TBBz and DMAT was determined comparing 2D gel spot using ANOVA (p = 0.003) and comparison of treatment means was done by T-test at p = 0.05. (C) Representative MALDI MS spectrum of trypsin-digested peptides picked from spot 3 and corresponding to QR2 (MW 25936, pi 5.88). (D) MS/MS spectrum of the 1986.10 Da peptide fragment derived from QR2 (spot 3). (E) Peptide match summary for QR2 as identified by MASCOT. Matched peptides and 46% sequence coverage are highlighted in red. 65

ATP-competitive inhibitors including the structurally-related compounds TBB, TBBz and DMAT (Figure 2-1). However, the molecular mechanism by which these chemicals function in cells has not been systematically explored. An unbiased characterization of available CK2 inhibitors was undertaken to investigate CK2 as a pharmacological anti-cancer target, as well as to explore the potential of the inhibitors to study CK2 function. The inhibitors displayed varying abilities to induce apoptosis in cells suggesting a distinct mechanism of action. Validation of the inhibitor's ability to reduce CK2 activity in cells was demonstrated by the loss of autophosphorylation of CK2|3 by CK2a/a', which was then restored in cells expressing inhibitor-resistant CK2 mutants. Rescue experiments, addressing inhibitor specificity were carried out and determined that the apoptosis observed following DMAT and TBBz treatment could not be rescued by reintroduction of functional CK2. To validate CK2 as an inhibitor target, a chemo-proteomic approach was carried out, which determined that CK2a and CK2a' were indeed TBB, TBBz and DMAT targets. Significantly, a number of putative off-target drug interactions were identified, including a novel TBBz and DMAT target, QR2. The discovery of QR2 as a putative CK2 inhibitor drug target provides possible explanations for the toxic effects associated with the inhibitors, as well as raises the prospect of a new therapeutic potential for TBBz and DMAT in other diseases. The unbiased evaluation of CK2 inhibitors provides evidence that TBBz and DMAT have potential off-target effects; however, due to the prominent role of CK2 in tumorigenesis, inhibition of this kinase remains a promising anti-cancer therapeutic avenue. The classical method for addressing kinase inhibitor specificity has been the use of panel screens, where a number of kinases from different families are tested for an inhibitory effect [3]. The limitation to this strategy has become evident, as the screens are not comprehensive and fail to consider the presence of other ATP-binding proteins, including key metabolic and regulatory proteins. The chemo-proteomic approach comprises an unbiased identification of drug binding proteins through ATP affinity purification in conjunction with proteomics and mass spectrometry [33]. A chemo- proteomic approach revealed that the CK2 inhibitors prevented CK2a and CK2a' from binding to ATP-Sepharose in a concentration-dependent manner, reiterating that CK2 was 66 a target of TBB, TBBz and DMAT. Significantly, the observation that 1 uM TBBz was not a sufficient concentration to inhibit CK2a' from binding to the ATP-Sepharose but was, however, an adequate concentration to induce apoptosis in HeLa cells suggested that the apoptosis was not due to the inhibition of CK2. Therefore, further analysis was performed to identify off-target protein-inhibitor interactions that could provide an explanation for the inability of inhibitor-resistant CK2 mutants to rescue the apoptosis associated with TBBz and DMAT. An analysis of the ATP binding proteins treated with CK2 inhibitors revealed a number of interesting proteins involved in cell survival, metabolism and drug detoxification (Table 2-1). Competition of Quinone Reductase 2 (QR2) from binding to ATP-Sepharose was observed with TBBz and DMAT but not TBB, indicating drug target differences between inhibitor derivatives. QR2 has a role in the detoxification processes of quinones, specifically, the two-electron reduction of menadione by the oxidation of N-alkylated or N-ribosylated nicotinamides [34]. Knockout models of QR2 in mice result in an increased susceptibility to polycyclic aromatic hydrocarbon-induced skin carcinogenesis, while inhibition of QR2 using quinacrine resulted in death of malaria-infected red blood cells by altering the redox status of the cell [33, 35]. Recently, a role for QR2 in the TNF apoptotic signaling pathway has emerged, where QR2 deficient keratinocytes where shown to have suppressed survival signals and increased TNF-induced apoptosis [36]. Intriguingly, other studies investigating the mechanism of action of ABL and PKC kinase inhibitors, demonstrated QR2 as a nonkinase novel drug target, suggesting a potential role for QR2 in the drug metabolism [37-39]. In the present study, the discovery of QR2 as a novel TBBz and DMAT drug target provides opportunities to further explore the mechanism by which cell viability is affected following treatment with CK2 inhibitors. Interestingly, Quercetin, a flavanoid and known inhibitor of CK2 with an IC50 = 0.55 [iM has also been described as a potent competitive inhibitor of QR2 with a Kj = 0.021 uM [40, 41]. The crystal structure of QR2 with the inhibitor resveratrol has been solved and showed that the active binding site was composed of a very hydrophobic cavity favoring the binding of flat planar conformations [40]. The hydrophobic cavity of QR2 could facilitate the binding of CK2 inhibitors which were designed to maximize hydrophobic interactions with the large bulky side chains within the CK2 ATP binding pocket. The 67 evolution of TBB to generate higher affinity inhibitors for CK2 resulted in an imidazole ring substitution, as well as the addition of bulky constituents, to facilitate the interaction with the hydrophobic pocket of CK2 [19]. One could envisage the strong hydrophobic binding properties and planar structure of TBBz and DMAT binding into the QR2 hydrophobic cavity, potentially altering or inhibiting its ability to function. Previous studies have shown that QR2 has the potential to activate CB1954 prodrug into a highly toxic DNA damaging agent, indicating the possibility that TBBz and DMAT could be modified [42]. The inability of TBB to bind to QR2 offers an explanation as to why TBBz and DMAT are more potent at inducing apoptosis. TBBz and DMAT could bind to QR2 and either inhibit the function of the protein leading to alterations in redox levels, or could potentially be modified by QR2 into more cytotoxic compounds facilitating an off- target response. However, further inhibitory studies are required to test the efficacy of TBBz and DMAT to inhibit QR2 in vitro as well as in vivo. It will be of interest to determine the efficiency of TBBz and DMAT to inhibit QR2, as they might present as novel inhibitors for other diseases such as anti-malarial research. The use of the inhibitors to study the role of CK2 in physiological processes will require caution and the use of rescue experiments to validate the CK2-dependent phenotypes. The identification of a number of putative metabolic off-targets provides evidence that the study of ATP-competitive inhibitors such as CK2 inhibitors will require vigorous testing to validate the specificity of the inhibitors in cells. Further, the relative abundance of the proposed drug target within various cells will also be essential in determining the specificity of inhibitors. Other cellular targets that are less sensitive to the inhibitors may be affected or inhibited by the drugs, due to the higher expression than the proposed target. Therefore, other strategies to inhibit CK2 may be required to maximize selectivity, including identifying compounds that inhibit CK2 via allosteric interaction, independent of the ATP-binding site. The production of peptide inhibitors specific for CK2 has been a promising avenue of research, as a number of groups have successfully developed and characterized the inhibition of CK2 in cells. Inhibition of CK2 using a CK2-specific peptide resulted in the reduction CK2 activity and of tumor growth in a mouse model, reiterating CK2 as a promising drug target [43]. 68

2.5 References

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CHAPTER 3 DECIPHERING CELLULAR DESCISIONS OF LIFE AND DEATH: CONVERGENCE OF PROTEIN KINASES AND CASPASE SIGNALING

3.1 Introduction

Apoptosis is an essential biological process required for normal cell turnover, proper embryonic development, as well as chemically induced cell death [1]. Furthermore, perturbations in the regulation of apoptosis signaling pathways have been associated with many human diseases, including cancer [2]. The progression of apoptosis is dependent on the tightly-regulated proteoyltic cleavage of a variety of proteins required for cell survival by a group of cysteine-dependent aspartic-directed proteases called caspases [1, 2]. Studies investigating the sequence requirements for caspase substrate recognition identified a stringent specificity for an aspartic acid residue within the cleavage site [3, 4]. Notably, the presence of charged or bulky residues adjacent to the Asp residue of position PI' are poorly tolerated in caspase catalysis, where the smaller residues Gly, Ala, Thr, Ser or Asn are preferred [4]. For example, phosphorylation of a Ser residue at the PI' site adjacent to the caspase cleavage site has been demonstrated to abolish proteolysis of caspase target peptides, indicating that the addition of a large phosphate sterically hinders the ability of the aspartic protease to cleave at the adjacent residue [5].

Recently, a role for protein kinases in the regulation of caspase signaling pathways has emerged, where the phosphorylation of caspases and/or caspase substrates has been shown to negatively influence caspase-mediated cleavage [5, 6]. The phosphorylation of caspase-9 by ERK [7], PKCt [8], c-Abl [9], CK2 [10] and Akt [11] has been shown to influence caspase-9 activity, illustrating the convergence of multiple protein kinases in the regulation of caspase signaling pathways. Interestingly, phosphorylation of caspase substrates by protein kinases at or near the caspase cleavage site has been shown to prevent caspase-mediated cleavage, signifying a potential mechanism for the regulation of caspase activity. Phosphorylation of Nogo-B within a 74 caspase recognition motif by CDK1 and 2 prevented its caspase-7-mediated cleavage, while caspase-3 -mediated cleavage of Calsenilin was shown to be regulated by CK1 phosphorylation [12, 13]. CK2 (formally known as, Casein Kinase II) is a serine/threonine protein kinase that has been implicated in the regulation of key tumor suppressors and oncogenes within cell survival pathways, promoting tumorigenesis [14]. Mounting evidence suggests an anti-apoptotic role for CK2 via the protection of pro- survival proteins from caspase-mediated cleavage. Specifically, CK2 phosphorylation of caspase targets Bid [15], Max [16], HS1 [17], PSN-2 [18], Connexin 45.6 [19], Caspase-9 [10] and PTEN [20] at residues at or near the caspase cleavage site have been shown to prevent caspase cleavage leading to the protection of cells from apoptosis. CK2 is constitutively active, ubiquitously distributed protein kinase, implicated in tumorigenesis and transformation with a unique consensus sequence for phosphorylation Ser/Thr-X-X- Acidic, where the acidic residue is one of Asp, Glu, pSer or pTyr [21]. The requirement for acidic residues for both CK2 phosphorylation and caspase protease activity points towards a potentially widespread mechanism for the regulation of apoptosis, via the protection of caspase targets by CK2 phosphorylation.

In the present study, an investigation of the global role of phosphorylation in the regulation of caspase signaling pathways was carried out. To test this mechanism of regulation, a comprehensive evaluation of overlapping CK2 and caspase targets was performed using a sequence-based Protein Identification Targeting Strategy, (sPITS). The strategy consisted of combining the global strengths of bioinformatics with the throughput capacities of peptide array target screens and the use of a novel method for identifying caspase targets, Caspase Substrate Identification (CSI) (Figure 3-1). Over 300 proteins with a CK2 phosphorylation consensus site at the P2 or PI' position within a caspase recognition sequence were identified. A number of peptides corresponding to proteins containing an overlapping CK2/caspase consensus were shown to be phosphorylated in the CK2 peptide array target screens, as well as cleaved by caspases using CSI, representing novel candidate CK2/caspase targets. Of particular interest, was the identification of an overlapping CK2/caspase motif within the caspase proteolytic activation site of pro-caspase-3. Evidence that phosphorylation at the P2 or PI' residues within the caspase cleavage recognition motif is sufficient to prevent caspase cleavage, as 75

well as the identification of numerous candidate CK2/caspase targets in the present studies, support a role for phosphorylation as a global mechanism of regulation of caspase signaling pathways.

Bioinformatics Phosphorylation Cleavage global identification of predicted targets by CK2 of targets by caspase

(Peptide match program) (Peptide arrays) (Caspase Substrate Identification)

CK2 targets (S/TxxD/E) DExD[S/T]xx[D/Ej

DE[S/T]Dx[D/E] H li I • I • 7 s Overlapping CK2 and Caspase tiiiMHBi recognition motif

Phosphorylation of peptide by CK2 at PI' and/ X Overlapping target l/V/LExD[S/Tlxx[D/E] or P2 prevents caspase Caspase targets peptides l/V/U:[S/T]Dx[D/E] mediated-cleavage 3,7 (DExD) phosphorylated by CK2 8,9 (1/V/LExD) Database Search

Phospho-ELM 31 CASBAH

Figure 3-1. Schematic of sPITS: a novel strategy used to identify global overlapping protein kinase and protease targets. A peptide match program was designed to comprehensively identify CK2/caspase targets within the human proteome that possess overlapping CK2 phosphorylation and caspase recognition motifs. Phosphorylation of the predicted targets by CK2 was investigated using peptide array target screens, while cleavage of candidate peptides by caspases was assessed using a novel throughput fluorescent cleavage assay, Caspase Substrate Identification (CSI). Evaluation of pre existing phosphorylation and/or caspase substrate databases was carried out to identify previously reported phosphorylation or caspase cleavage in cells. Protection of peptides predicted to be phosphorylated at the P2 and/or PI' residue by CK2 from caspase- mediated cleavage were examined using phosphorylated peptides in CSI assays. 76

3.2 Materials and Methods

3.2.1 Identification of overlapping CK2 and caspase targets using bioinformatics

A peptide match program was designed to search the human proteome databases (SwissProt and NCBI) for overlapping CK2 consensus and caspase recognition sequences. The program was engineered to search for peptides matching overlapping CK2 and caspase-3/7 sequences (D-E-[S/T]-D-X-[D/E\), (D-E-X-D-[S/T\-X-X-[D/E\) or CK2 and caspase-8/9 sequences ([I/V/L]-E-X-[S/T\-D-X-[D/E]), ([I/V/L]-E-X-D-[S/T\- X-X-[D/E]) where the CK2 phosphorylation site was at the P2 or PI' relative to the predicted Asp caspase cleavage site. Following a proteome-wide search for matching peptides, the peptide match program returned the GI number, SwissProt primary accession number and the SwissProt protein ID, as well as the matching peptide sequence (+/- 10 residues on either side of recognized peptide pattern). The biological process of the matched peptides was then assessed and organized based on their GO functions. Matched peptides were also imported into Phospho-ELM (http://phospho.elm.eu.Org/X Phosida (http://www.phosida.com/) and CASBAH (http://bioinf.gen.tcd.ie/casbah/) databases to identify proteins previously reported as being phosphorylated at the predicted CK2 site or cleaved by caspases.

3.2.2 Phosphorylation of peptide arrays by CK2

Peptide arrays were synthesized via an in situ SPOT technology on nitrocellulose (Intavis) using the Auto-Spot Robot ASP 222 (Amimed) [22, 23]. Peptides were briefly soaked in ethanol, washed in kinase assay buffer (50 mM Tris/HCl pH 7.5, 30 mM MgCk, 50 mM KC1,1 mM DTT) followed by incubation in kinase assay buffer overnight at room temperature with gentle agitation. The membrane was then incubated for 1 hr at 30 °C in fresh kinase assay buffer with gentle agitation. The kinase assay was initiated by addition of 10 mM ATP, 20 ^Ci [y32P]-ATP and 130 units of N-terminal GST-tagged domain of CK2 (GST-CK2a) and incubated for 20 min at 30°C (1 unit of activity of CK2 is defined by the amount of [y32P] incorporation (pmol/min) of into the standard peptide substrate RRREEETEEEEEE at 30°C). Kinase reactions were terminated via 10 washes 77 with 1M NaCl and 10 washes with dcfflbO, followed by incubation with stripping solution (4 M guanidine hydrochloride, 0.5% b-mercaptoethanol) for 1 hr at 40 °C with gentle agitation to decrease background on the array. The membrane was then washed multiple times in water, incubated with ethanol and dried. Phosphorylation of peptides by CK2 was visualized using the Phosphorimager (Molecular Dynamics), while relative [y32P] levels of each peptide were determined using ImageQuant TL software (Amersham Biosciences). Phosphorylation of peptide arrays were performed in triplicate, with a number of positive controls including the standard CK2 peptide substrate RRREEETEEEEEE as well as multiple negative peptide controls including RRREEEAEEEEEE with an Ala substitution for the Thr.

3.2.3 Caspase Substrate Identification (CSI)

Peptides identified from a proteome-wide bioinformatics search for overlapping CK2 consensus and caspase cleavage sites were synthesized on-resin by standard Fmoc- based peptide chemistry using an Automated Multiple Peptide Synthesizer (Intavis). NHS-Fluorescein (FL) (Pierce) was labeled on the N-terminus of the peptides according to the manufacturer's manual before TFA cleavage, while Fmoc-Lys(Biotinyl)-OH (BACHEM) was attached to the C-terminus. Phosphorylated peptides were generated by the addition of Fmoc-Thr[PO(OBzl)-OH]-OH (AnaSpec) or Fmoc-Ser[Ser[PO(OBzl)- OHJ-OH (AnaSpec). Phosphorylated and non-phosphorylated peptides (13 amino acids in length at a concentration of 600 pmol) were incubated with 6U/uL active purified caspase-3 (One U-lpmol/min at 30 °C using Ac-DEVD-pNA, BIOMOL catalog # P-412) in caspase assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM DTT) for 1 hr at 30 °C in 96-well plates (Evergreen Scientific). Following the cleavage assay, peptides were then transferred to streptavidin high capacity-coated 96 well plates (Sigma) and incubated for 2 hrs at 30°C. Following incubation, plates were washed thoroughly to remove unbound peptides and NHS- fluorescein fluorescence was detected via the Victor 3V system (Perkin Elmer) using an excitation wavelength of 485 nm and emission wavelength of 535 nm ('parameter' = 0.1 s). 78

3.2.4 Mass spectrometry analysis of peptide cleavage

Peptides were incubated with caspase-3 in caspase assay buffer for 1 hr at 30 °C with gentle agitation. Following incubation, samples were spotted on a MALDI plate (Micromass, Manchester, UK), and cleavage of the peptides by caspase-3 was assessed via analysis of the peptide products observed in the mass spectra. Mass spectra were acquired on a M@LDI-LR (Micromass, Manchester, UK) mass spectrometer in positive reflectron mode with time-of-flight (TOF) detection. The instrument was calibrated with PEG1000, PEG2000, PEG3000 and Nal and peptide masses are quoted relative to an angiotensin internal standard. Spectra were collected between an m/z range of 800 to 2600 and analysis of the spectra was performed using MassLynx 3.5 software.

3.2.5 Caspase-3 activity assay

To assess the relative caspase-3 activity levels for each peptide treatment in the in vitro fluorescent peptide cleavage assay, the cleavage of the ideal caspase-3 consensus sequence (Ac-DEVD-pNA) (P412) (BIOMOL) was measured following fluorescein detection to validate activity. Peptides were incubated with purified caspase-3 in caspase assay buffer with 200 mM Ac-DEVD-pNA substrate in 96-well plates (Evergreen Scientific) and continuously monitored at A^snm via the Victor 3V system (Perkin Elmer). All treatments were performed in triplicate and the amount of caspase activity (pmol/min/mg) for each peptide treatment was determined.

3.3 Results

The protection of key pro-survival proteins from caspase-mediated cleavage by CK2 phosphorylation has implicated a role for CK2 in the direct regulation of caspase signaling. To investigate whether phosphorylation of targets within the caspase cleavage site was a global mechanism for protection of cells from apoptosis, a large-scale proteome-wide bioinformatics search for proteins possessing an overlapping CK2 consensus motif for phosphorylation and caspase cleavage recognition sequence was undertaken. Specifically, protein sequences were targeted that contained a CK2 phosphorylation consensus site at the P2 or PI' position adjacent to the Asp residue cleaved by caspase-3, -7, -8 and -9. Previously, studies investigating caspase cleavage 79 demonstrated that peptides phosphorylated at a Ser in the PI' position prevented proteolysis by caspases [5, 16]. The addition of the bulky phospho-group sterically prevented the ability of caspase to hydrolyze the Asp-Pi' scissile bond, implicating a novel mechanism by which proteins could be regulated during apoptosis. In the present study, a peptide match program was engineered to identify protein sequences from the human proteome database (Swiss-Prot) that contained overlapping CK2/caspase recognition motifs. Bioinformatics analysis revealed over 300 putative CK2/caspase targets with a [Ser/Thr] CK2 site at the P2 or PI' position within the caspase-3, -7, -8 or - 9 recognition sequence (Appendix A, Table A-l). Of particular interest, were a number of proteins identified in the bioinformatics search that had been previously reported to be cleaved by caspase at an overlapping CK2/caspase site, including pro-caspase-3 and pro- caspase-8 (Table 3-1).

Table 3-1. Proteins identified in peptide match search with an overlapping CK2 and caspase recognition motif cleaved by caspases in cells.

Name Protein ID Sequence Caspase Apoptosis regulator BAX, membrane BAXA_HUMAN klseclkrigDELDSNMElqrmiaav unknown isoform alpha* Eukaryotic translation IF4H_HUMAN kfkgfcyvefDEVDSLKEaltydgal 3 initiation factor 4H Caspase-3 precursor* CASP3_HUMAN acrgteldcglETDSGVDddmachk 8/9 DNA-binding protein SATB1_HUMAN khfkktkdmmVEMDSLSElsqqg 6 SATB1 Protein Max MAX_HUMAN msdnddlEVESDEEqprfqsaadk 5 Caspase-8 precursor* CASP8_HUMAN qgdnyqkgipVETDSEeqpylemd 8 PH and SEC7 domain-containing PSD4 HUMAN wtldasqsslLETDGEqpsslkkkea unknown protein 4 * Protein essential in the progression of apoptosis

3.3.1 Functional characterization of overlapping CK2/caspase targets

Following identification of the putative CK2/caspase targets from the bioinformatics screen, further data analysis was carried out to classify these proteins with respect to their GO function (Gene Ontology). The most prevalent functions were found to be DNA/RNA-related proteins (23 %), proteins involved in metabolism (19.5 %), 80

Signaling (16 %) as well as in cell death differentiation (13.5 %) (Figure 3-2A). To determine whether the putative CK2/caspase targets were phosphorylated at the predicted CK2 site in cells, a comprehensive evaluation of phospho-databases, including Phosida and Phospho-ELM was performed. Intriguingly, 37 proteins (11%) identified in the bioinformatics search are phosphorylated at the predicted [Ser/Thr] site in cells, while 87% have not been characterized, providing further evidence for a global role for CK2 in the protection of targets from caspase-mediated cleavage (Figure 3-2B). Further analysis of the protein kinases responsible for the phosphorylation of the putative CK2/caspase targets revealed that only 6 of the 37 (16 %) have been identified, 5 of which were shown previously to be phosphorylated by CK2 in cells (Table 3-2). The remaining 85%) of the putative CK2/caspase targets phosphorylated in cells have not been characterized, reiterating the importance of identifying the functional role of CK2 in these phosphorylation events.

3.3.2 Phosphorylation of putative targets on peptide arrays by CK2

To comprehensively evaluate whether the CK2/caspase target sequences identified from the bioinformatics screen were phosphorylated by CK2, a peptide array target screen was performed. Peptide arrays were synthesized encompassing all the identified protein sequences from the bioinformatics screen that possessed an overlapping CK2 consensus motif and caspase recognition sequence. Incubation of the peptide array membranes with GST-CK2a in the presence of [y32P]-ATP showed that a number of peptides were phosphorylated, as evident by the presence of a [y32P] labeled spot (Figure 3-2C). Relative [y32P] incorporation into peptides was determined using ImageQuant LR software where peptides with [y P] readings 3-fold greater than the negative control (RRREEEAEEEEEE) were reported as being phosphorylated by CK2. Phosphorylation of control peptides corresponding to Max, BID, PTEN and Presenilin-2 (PSN2) in the peptide array target screens validate that the target screens can efficiently identify previously reported CK2/caspase substrates. Notably, the phosphorylation of peptides corresponding to other previously reported caspase substrates; pro-caspase-3, pro- caspase-8, Bax and Eukaryotic translation initiation factor 4H were shown to be phosphorylated by GST-CK2a in the peptide arrays (Figure 3-2D). A complete list of 81

Various prote!n

Proteins „ a"d„ „ „, Degradation (2%) ,z%' (10%) (11%) • Cleaved by caspase

• Phosphorylated at predicted Signaling (16 %) CK2 site n unknown

*« * *» « » t - *••»•••»• •* •- ^ •> • * • m • * *

Max: IEVESDEE BID: LQTDGNRS PTEN: DVSDNE EIF4H: DEVDSLKE SATB1:VEMDSLSE PSN2: DSYDSFGE Bax: DELDSNME pro-caspase-3: IET0SGVD pro-caspase-8: VETDSEEQ

20000 40000 60000 80000 32P incorporation

Figure 3-2. Functional characterization of proteins identified with an overlapping CK2/caspase consensus sequence. (A) Biological process of candidate targets was characterized based on their GO function (Gene Ontology). (B) Comprehensive database searches were performed to identify phosphorylated candidate CK2/caspase in cells using Phospho-ELM and Phosida, while caspase substrates were evaluated using CASBAH database. (C) A representative CK2 peptide array of proteins identified in the bioinformatics search that contain an overlapping CK2/caspase sequence. Peptide arrays containing the putative CK2/caspase target consensus sequences were incubated with GST-CK2oc and relative [y- P] incorporation of into peptides was visualized on a phosphorimager and determined using ImageQuant LR software. Peptide arrays were performed in triplicates. (D) Proteins identified to be phosphorylated by GST-CK2a in the peptide arrays that are known caspase substrates. 82

Table 3-2. Proteins identified from peptide match screen with an overlapping CK2 and caspase recognition motif previously reported to be phosphorylated in cells. Phospho- Elm and phosida databases were analyzed.

Swiss-Prot ID Sequence Kinase MPP10_HUMAN sdeditnvhdDELDSNKEddeiaeeeae unknown UB2R1HUMAN eeeadscfgdDEDD SGTEe CK2 CHD8_HUMAN DEDDSDSEldls unknown NFIA_HUMAN sstkrlksveDEMD SPGEepfytgqgrs unknown CV019_HUMAN lfkppedsqdDESDSDAEeeqttkrrrp unknown NOLCl_HUMAN qpvessedssDESDSSSEeekkpptkav unknown VPS72JHUMAN eyqgdqsdteDEVDSDFDidegdepssd CK2 NOL8_HUMAN sgklfdssddDESDSEDDsnrfkikpqf unknown BAZ1BHUMAN rkkfpdrlaeDEGDSEPEavgqsrgrrq unknown VAMP4_HUMAN gsvkserrnlLEDDSDEEedfflrgpsg unknown NUCL_HUMAN pkkmapppkeVEEDSEDEemsedeedds unknown RBL2_HUMAN pasttrrrlfVENDSPSDggtpgrmppq CDK2 CO039_HUMAN gpvmygklprLETDSGLEhslphsvgnq unknown NOL8_HUMAN ddrfrmdsrfLETDSEEEqeevnekkta unknown MY18AHUMAN vtkyqkrknkLEGDSDVDseledrvdgv unknown AVEN_HUMAN ggwgagasapVEDDSDAEtygeendeqg unknown SDA1_HUMAN apgkcqkrkylEIDSDEEprgellslrd unknown MBD2_HUMAN sraadteemdIEMDSGDE unknown RRP15_HUMAN kdhfysdddalEADSEGDaepcdkenen unknown DDEF2_HUMAN yewrllhedlDESDDDmdeklqpspn unknown MRE11JHUMAN tknysevievDESDVEedifpttskt CK2 CV019_HUMAN lfkppedsqdDESDSDaeeeqttkrr unknown NIPBL_HUMAN knntaaetedDESDGEdrgggtsgsl unknown NOL8_HUMAN sgklfdssddDESDSEddsnrfkikp unknown CPSF2_HUMAN qskeadidssDESDIEedidqpsahk unknown MAN1_HUMAN gskvllgfssDESDVEasprdqaggg unknown MPP10_HUMAN nlkykdffdpVESDEDitnvhddeld unknown L1CAM_HUMAN kdetfgeyrsLESDNEekafgssqps CK2 MAXHUMAN msdnddieVESDEEqprfqsaadk CK2 CHD4_HUMAN rsssedddldVESDFDdasinsysvs unknown N0L8_HUMAN ddrfrmdsrfLETDSEeeqeevnekk unknown CA052_HUMAN arllpegeetLESDDEkdehtskkrk unknown SETD2_HUMAN rgplkkrrqelESDSEsdgelqdrkk unknown WDR70_HUMAN ktqpktmfaqVESDDEeaknepewkk unknown CBPD_MOUSE kksllshefqDETDTEeetlyssk unknown CTNA2M0USE avlmirtpeeLEDDSDFEqedydvrsrt unknown ENSP00000307525 tipVESDDDegap unknown SF3A1_HUMAN fgeseevemeVESDEEddkqekaeep unknown 83 candidate CK2/caspase targets phosphorylated by GST-CK2ct can be found in supplementary materials (Appendix A, Table A-2).

3.3.3 Identification ofcaspase targets using Caspase Substrate Identification (CSI)

Further studies to determine whether the candidate CK2/caspase peptides phosphorylated by CK2 in the peptide arrays were also cleaved by caspases were carried out using a fluorescent-based cleavage strategies. An in vitro fluorescent peptide cleavage assay was engineered, Caspase Substrate Identification (CSI), which was capable of detecting caspase cleavage of a large number of putative CK2/caspase targets simultaneously (Figure 3-3). Firstly, peptides containing a caspase consensus motif, demonstrated to be phosphorylated by CK2 in the peptide array assays were synthesized with a Fluorescein (FL) N-terminal label and a C-terminal Biotin label (FL/Biotin peptide). (2) Phosphorylated and the corresponding non-phosphorylated FL/Biotin labeled peptides were then incubated with the appropriate active caspase-3 or caspase-8, caspase inactive mutants or no caspase treatment in 96-well plates. (3) Cleavage of the peptides by caspase within the caspase motif resulted in the generation of two proteolytic fragments, an N-terminal FL-labeled cleavage product and a C-terminal-biotin labeled peptide fragment. Only the uncleaved peptides, containing both the fluorescein and biotin label were detected using CSI. (4) Cleavage of the FL/Biotin labeled peptides by active caspase was then determined by the quantification of fluorescein counts/0.Is bound to Streptavidin-coated 96 well plates using an automated Victor 3V system plate reader (Perkin Elmer) as described in material and methods. A statistical significant difference using ANOVA (P< 0.01) in fluorescein counts/0.Is bound to Streptavidin-coated plates between the active caspase-3 treatment and the control inactive pro-caspase-3 (CI63A) or no caspase treatment indicated the peptide was cleaved by caspase.

3.3.4 Validation of caspase-mediated cleavage using mass spectrometry in conjunction with CSI

To validate the use of CS7for the detection of caspase-mediated cleavage, a mass- spectrometry based evaluation of the cleavage of FL/Biotin labeled peptides by caspases was carried out. Addition of caspase-3 to the FL/Biotin-labeled peptide corresponding to 84

- Caspase cleavage site V /--v . Incubation of peptide and corresponding phospho-peptide with active caspase v rl- JSfM AWiR

^ V Loss of N- Incubation with terminal streptavidin coated 96 I—e^f" Biotin fluorescein label well plates upon cleavage

fiiidrescein counts bound to streptavidin- coated plates determined via fluorescent plate reader

Peptides protected from cleavage will be positive for fluorescein signal Cleavage No cleavage

Figure 3-3. Caspase Substrate Identification (CSI), a novel method to detect caspase cleavage. [1] Peptides representing CK2/caspase candidate targets were synthesized with an N-terminal fluorescein label and C-terminal Biotin label and incubated with active or inactive caspase. [2] Following caspase cleavage, the peptides were incubated with [3] streptavidin-coated-plates and the fluorescein counts/0.Is was determined [4]. A statistical analysis of the fluorescein bound counts between the active and inactive caspase treatments was determined using ANOVA (P< 0.01). 85 the positive control PARP caspase-3 cleavage sequence (DEVD) resulted in the generation of an N-terminal FL-labeled proteolytic fragment (FL-RKGDEVD, m/z = 1176.46) and C-terminal Biotin-labeled fragment (GVDEVLK-Biotin, m/z = 1006.51) confirming that caspase-3 could efficiently cleave the peptides in vitro (Figure 3-4A). Notably, the efficiency of caspase-3 to cleave the FL/Biotin PARP-peptide was quite high, as evident by the complete loss of the full-length peptide (FL- RKGDEVDGVDEVLK-Biotin, m/z = 2143.85) in the mass spectrum. Following confirmation by mass spectrometry, that caspase-3 could efficiently cleave FL/Biotin labeled peptides in solution, an evaluation of the effectiveness of CSI to detect caspase- mediated cleavage was carried out. Labeled peptides corresponding to the caspase-3 PARP cleavage sequence were treated with active caspase-3 or inactive caspase-3, followed by incubation with Streptavidin-coated plates. A statistical difference in fluorescein counts/0.Is bound to Streptavidin was observed via ANOVA analysis (P <0.01) between treatment of FL/Biotin-peptides with active caspase-3 and inactive caspase-3, indicating that caspase-mediated cleavage occurred resulting in the loss of the FL-labeled N-terminal proteolyic fragment (Figure 3-4B). The diminished fluorescein counts/0.Is bound to streptavidin following treatment of FL/Biotin-peptides by active caspase-3 was consistent with the loss of the N-terminal FL-labeled protolytic fragment observed in the mass spectrometry analysis, demonstrating that CSI could efficiency be utilized to detect protease activity. To investigate the effect of phosphorylation of the P2 and PI' residues on caspase-mediated peptide proteolysis, phosphorylated FL/Biotin- peptides were synthesized. Phosphorylated FL/Biotin-labeled mutant PARP (DETDS) peptides at the P2 (DETpD) or the PI' (DEVDSp) residue were incubated with either active or inactive caspase-3, followed by the assessment of cleavage using CSI. Non- phosphorylated mutant PARP (DETDS) peptides were shown to be cleaved in mass spectrometry and CSI analysis, demonstrating that mutation of the PARP sequence did not interfere with caspase recognition or caspase-mediated cleavage (Figure 3-4C and D). Protection of phosphorylated FL/Biotin-peptides from caspase-mediated cleavage was observed, where phosphorylation of the P2 or PI' residues of the mutant PARP (DETDS) peptides resulted in no statistical difference in fluorescein counts/0.Is bound to Streptavidin between active caspase and inactive caspase treatments (Figure 3-4E). 86

2143.15 ©o 100 100

OQ9 looe.sij

U*P m/i 1000 1500 2000 o iooo" 1500 5ooo '2W OEVDG + DEVDG* RKGDEVDGVDEVLK RKGDEVDGVDEVLK Caspase-3 caspase-3 + Caspase-3 + Caspase-3 (inactive) (inactive)

100 2232.82 100 177«.41

QIC 1093.52 1296.69 I /n/i M r- 1000 1500 200o' 2500 1000 1500 2000 2500 DETDS* DETDS* RKGDETDSGVDEVLK RKGDETDSGVDEVLK Caspase-3 Caspase-3 (inactive) + Caspase-3 + Caspase-3 (inactive)

DETpDG • DETpDG + DEVDSp + DEVDSp* Caspase-3 caspase-3 Caspase-3 caspase-3 (inactive) (inactive)

Figure 3-4. Validation of CSI to detect caspase cleavage. (A) N-terminal fluorescein labeled and C-terminal Biotin peptides *tsm corresponding to the caspase-3 PARP cleavage motif were treated with active or inactive caspase-3 and cleavage was detected using MALDI TOF mass spectrometry. Following caspase-3 treatment, an analysis of N-terminal fluorescein labeled •^and the C-terminal Biotin labeled 9m proteolytic cleavage products was assessed using mass spectrometry. (B) In comparison, caspase-3 cleavage of the PARP peptides was then analyzed using CSI. The statistical difference in fluorescein counts/0.Is bound to streptavidin-coated plates was determined for peptides treated with or without active caspase-3. (C) N-terminal fluorescein labeled and C- terminal Biotin peptides •saW corresponding to an engineered PARP (DETDS) sequence were treated with active or inactive caspase-3 and cleavage was detected using MALDI TOF mass spectrometry. (D) In comparison, PARP (DETDS) peptides were then analyzed for cleavage using CSI. (E) Protection of the phosphorylated PARP (DETpDS) and (DEVDSp) peptides at the P2 and PI' residues was determined using CSI. 87

Overall, these experiments validate the use of CSI in both the detection of protease activity, as well as in the identification of novel caspase substrates.

3.3.5 Global identification of candidate CK2/caspase targets sensitive to caspase cleavage

To exploit the use of CSI, an evaluation of CK2/caspase peptides identified in the bioinformatics search that were phosphorylated by CK2 in peptide array screens were analyzed for sensitivity to caspase-mediated cleavage. Specifically, candidate peptides that had previously been reported to be phosphorylated at the predicted overlapping CK2/caspase site in cells were analyzed for caspase-3 and/or 8 cleavage using CSI (Table 3-3). Notably, all of the known caspase substrates that were tested using CSI, including PARP1 and Fodrin were shown to be cleaved, demonstrating the efficacy of CSI to detect caspase cleavage (Figure 3-5A). Peptides corresponding to PTEN and PSN-2, which are known examples where phosphorylation of the PI' residue by CK2 results in protection from caspase-3 cleavage were also shown to be cleaved using CSI (Figure 3-5B). Interestingly, peptides corresponding to the proteolytic activation site of pro-caspase-3 were demonstrated to be cleaved by caspase-8 in the CSI assays, while a number of peptides were cleaved by caspase-3, including peptides corresponding to proteins involved in transcriptional regulation, Transcription factor-like 1 (YL-1), Chromodomain helicase DNA binding protein 8 (CHD8), Zinc finger and BTB domain containing 45 (Figure 3-6). A peptide corresponding to the PARP sequence with the Asp residues of the caspase-3 consensus sequence mutated to Ala was used as a control, which showed no caspase cleavage. Demonstration that CSI was capable of identifying multiple known caspase substrates, as well as a number of novel caspase sequences sensitive to caspase cleavage, illustrates the effectiveness of GST to assay proteolytic cleavage. 88

Table 3-3. Identification of candidate CK2/caspase peptide sequences sensitive to caspase-mediated cleavage. Cleavage of peptides by caspase was determined using CSI.

Protein Name Protein ID Sequence Caspase Cleavage PARP* PARP1 HUMAN rkgDEVDGvdev 3 Yes Presenilin-2* PSN2 HUMAN meeDSYDSFGEps 3 Yes PTEN* PTEN HUMAN svtpDVSDNEpdh 3 Yes PTEN* PTEN HUMAN ysDTTDSDPEnep 3 Yes Fodrin* SPTA2 HUMAN mprDETDSktasp 3 Yes Bid+ BID HUMAN egydeLQTDGnrs 8 Yes Bid+ BID HUMAN egydeLQTpDGnrs 8 No Pro-caspase-3+ CASP3 HUMAN cglETDSGVDddm 3/8/9 Yes Pro-caspase-3+ CASP3 HUMAN cglETpDSGVDddm 3/8/9 No Pro-caspase-3+ CASP3_HUMAN cglETDSpGVDddm 3/8/9 No Serine/threonine-protein MST4_HUMAN hsdDESDSEgsds 3 No kinase MST4 Carboxypeptidase D CBPDHUMAN fqDETDTEEEtlyss 3 No precursor M-phase MPP10_HUMAN dDELDSNKEddei 3 Yes phosphoprotein 10 transcription factor-like 1 VPS72 HUMAN eDEVDSDFDideg 3 Yes nucleolar protein 8 NOL8 HUMAN dDESDSEDDsnrf 3 No THO complex 5 THOC5_HUMAN dDESDSDAEeeqt 3 No zinc finger and BTB domain ZBT45_HUMAN esdDETDGEdgeg 3 Yes containing 45 F-box only protein 3 isoform FBX3_HUMAN admDESDEDdeee 3 No 1 chromodomain helicase DNA CHD8_HUMAN DEDDSDSEldls 3 Yes binding protein 8 development- and differentiation-enhancing DDEF1_HUMAN edlDESDDDmdek 3 Yes factor 1 Serine/threonine-protein LMTK1_HUMAN edsDESDEElrcy 3 No kinase LMTK1 NKF3 kinase family member 148368962 eswDESDEEllam 3 No meiotic recombination 11 24234690 ievDESDVEedif 3 No homolog A isoform 2 interferon alpha/beta receptor 46488937 nydDESDSDteaa 3 No 2 isoform a NMD3 homolog 113419592 tipVESDDDegap 3 No nucleolar protein 8 NOL8_HUMAN srfLETDSEeeqe 3 No chromodomain helicase CHD4_HUMAN dldVESDFDdasi 3 No DNA binding protein 4 vesicle-associated VAMP4_HUMAN eLEDDSDFEqedy 3 No membrane protein 4 cell death regulator aven AVEN_HUMAN pVEDDSDAEtyge 3 echinoderm microtubule EMAL1_HUMAN shsDESDSDlsdv 3 Yes associated protein huntingtin interacting SETD2_HUMAN elESDSESDgelq 3 No protein B histone deacetylase 4 HDAC4 HUMAN qepIESDEEeaep 3 No Pro-caspase-8 CASP8_HUMAN grpVETDSEsefp 3 No nucleolar protein with N0M1_HUMAN dLESDSQDEseee 3 No MIF4G domain 1 Random control sequence, no CONTROL rkgAEVAGVdev 3 No caspase-3 site *Cleaved by caspase-3 in cells, + cleaved by caspase-8 in cells. 89

PARP1 FODRIN

6000 C (A '55 T 5000 o o to "-' V W I- *« o c 3000 3 3 = 3

RKGDEVDGVDEVLR RKGDEVDGVDEVLR+ mprDETDSktasp mprDETDSktasp C3 C3 B PTEN PSN-2

40000.00

35000.00

30000.00 '55 T o o ,25000.00 w 0) (ft 20000.00 i_ <•> o c 15000.00 3 3 IEEE c<3 10000.00 5000.00

0.00 ysDTTDSDPEnep ysDTTDSDPEnep meeDSYDSFGEps meeDSYDSFGEps C3 C3

Figure 3-5. Detection ofcaspase cleavage of known cellular caspase targets using CSI. (A) Peptides corresponding to PARP1 and Fodrin cleavage sites were incubated with or without caspase-3 and proteolysis by caspase-3 assayed using CSI. (B) Peptides corresponding to previously identified CK2/caspase targets PTEN and PSN2 were incubated with or without caspase-3 and cleavage by caspase-3 detected using CSI. A statistical difference in fluorescein counts (0.1s) bound to streptavidin was determined using ANOVA (p=0.01). All experiments were performed in triplicate. 90

VSP72 HUMAN MPP10 HUMAN

£ 25000

eDEVDSDFDideg eDEVDSDFDideg dDELDSNKEddei dDELDSNKEddei C3 C3

ZBT45 HUMAN CHD8 HUMAN

esdDETDGEdgeg esdDETDGEdgeg eDEDDSDSEIdls eDEDDSDSEIdls C3 C3

EMAL5 HUMAN DDEF2 HUMAN

C 10000

C 6000

shsDESDSDIsdv shsDESDSDIsdv edIDESDDDmdek edIDESDDDmdek C3 C3 MST4 HUMAN Random

16000 .£ 14000 jjj 12000 (B 10000 £ 8000 § 6000 j£ 4000 2000 0 hsdDESDSEgsds hsdDESDSEgsds RKGAEVAGVDEV RKGAEVAGVDEV C3 + C3

Figure 3-6. Identification of candidate CK2/caspase-3 targets using CSI. Peptides corresponding to proteins identified to contain an overlapping CK2 and caspase recognition motif were incubated with or without caspase-3 and cleavage by caspase-3 assayed using CSI. A statistical difference in fluorescein counts (0.1s) bound to streptavidin was determined using ANOVA (p=0.01). All experiments were performed in triplicate. 91

3.3.6 Phosphorylation of the P2 and PI' residue of caspase-3 peptides prevents caspase cleavage Identification of pro-caspase-3 as a candidate CK2/caspase substrate from the bioinformatics screen that was phosphorylated by CK2 in the peptide target screens, prompted further studies to determine the effect of phosphorylation at the P2 or PI' residues on caspase cleavage. Alignment of pro-caspase-3 across species revealed that the CK2 phosphorylation sites within the proteolytic activation site were highly conserved, suggesting a role for CK2 in the regulation of caspase-mediated cleavage (Figure 3-7A). FL/Biotin-labeled caspase-3 peptides containing the proteolytic activation site, phosphorylated at the P2 (IETpD) or the PI' (IETDSp) residue were incubated with either active or no caspase-8, followed by the assessment of cleavage using CSI. A statistical difference in flourescein counts/0.Is bound to Streptavidin between active and no caspase-8 treatments was observed in the non-phosphorylated caspase-3 peptides (Figure 3-7B). Protection of phosphorylated FL/Biotin-caspase-3 peptides from caspase-8- mediated cleavage was observed, where phosphorylation of the P2 or PI' residues within the proteolytic activation site resulted in no statistical difference in flourescein counts/0.Is bound to Streptavidin between active and no caspase-8 treatments (Figure 3- 7C). The resistance of the P2 and PI' phosphorylated peptides to caspase-8 cleavage suggests that phosphorylation on either side of the cleaved Asp was sufficient to prevent caspase proteolysis. These findings are consistent with previous studies demonstrating that the addition of bulky charged groups at the PI' position abolished caspase catalysis, supporting a role for phosphorylation as a mechanism for the negative regulation of caspase cleavage. Intriguingly, the phosphorylation of pro-caspase-3 at Thrl74 and Serl76 within the caspase-8 and -9 cleavage site would presumably prevent the activation of the key effector caspase in the execution phase of apoptosis, introducing a novel mechanism of regulation of caspase signaling.

3.4 Discussion

B P2 P1' 6000

Human GIETDSGVDD 180 5000

Rat GIETDSGTDD 180 4000 Mouse GIETDSGTDE 180 j2 3000 Dog GIETDSGIED 180 Pig GIETDSGTED 180 2000 Fish GIETDSG-ED 184 1000

IETDSG + IETDSG + no Caspase-8 Caspase-8

lETpDS + lETpDS + lETDSpG + lETDSpG + no caspase-8 Caspase-8 no-caspase-8 Caspase-8

Figure 3-7. Addition of a phosphate group at the P2 or PI' residue within the proteolytic activation site of pro-caspase-3 peptides prevents caspase-mediated cleavage. (A) Alignment of CK2 phosphorylation site in the pro-caspase-3 proteolytic activation site across species. (B) Cleavage of peptides corresponding to pro-caspase-3 by caspase-8 was detected using CSI. (C) Phosphorylated peptides at the P2 or PI' residues corresponding to the caspase-cleavage motif in caspase-3 were treated with or without caspase-8 and cleavage was assessed using CSI. All treatments were performed in triplicate; error bars represent differences in standard deviation 93

Specifically, phosphorylation of a residue at the PI' position adjacent to the cleaved aspartic residue has been shown to abolish caspase cleavage [5]. Based on crystal structure analysis of caspases bound to peptide substrates, it was proposed that addition of a large bulky charge phosphorylated group sterically prevented the caspase from hydrolyzing the PI' scissile bond [4, 5]. Evidence of a structural mechanism for phosphorylation-dependent protection of caspase substrates, as well as the protection of a wide variety of caspase targets by multiple protein kinases, suggests a global role for phosphorylation in the negative regulation of apoptosis. Protein kinase CK2, has been implicated in the protection of multiple caspase substrates from caspase-mediated cleavage, as well as shares an overlapping requirement with caspases for an acidic consensus sequences. Therefore, to investigate the global role of phosphorylation in caspase signaling, a comprehensive evaluation of overlapping proteins with a CK2 phosphorylation and a caspase recognition motif was carried out.

3.4.1 Development and exploitation of throughput strategies to study complex signaling networks

To elucidate the role of phosphorylation as a global mechanism in the negative regulation of caspase signaling, a throughput protein target identification strategy was required. In the present study, sPITS was employed that comprehensively identified protein targets based on overlapping protein kinase and caspase recognition consensus sequences. The approach consisted of combining the global strengths of bioinformatics with the throughput capacities of peptide array target screens and fluorescent cleavage assays. Following a proteome-wide search for CK2/caspase targets, over 300 proteins with a CK2 phosphorylation consensus site at the PI' or P2 position within a caspase recognition sequence were identified. A number of candidate CK2/caspase targets were shown to be both phosphorylated by CK2 in the peptide arrays and cleaved by caspase-3 using CSI, including proteins directly involved in caspase signaling and progression of apoptosis. In the present study, a novel strategy to identify caspase targets was engineered, which provided an efficient fluorescent assay capable of detecting caspase cleavage of multiple candidate CK2/caspase target peptides. Caspase Substrate Identification (CSI) was based on previous technologies used to detect caspase activity, 94 however, had the advantage of being able to test large numbers of candidate protein sequences for caspase sensitivity, simultaneously. CSI provides a novel technique for testing protease activity that has the capacity to be applied in high throughput studies. Exploitation of CSI to study protease substrate recognition, effect of post-translational modifications on proteolysis, as well as detection of protease activity from various sources will be highly advantageous. However, the identification of overlapping CK2/caspase substrates based on consensus does have the limitation of not detecting protein targets that do not conform to the general consensus sequences. Therefore, modifications to the parameters of the peptide match program to recognize a less stringent consensus specificity will be required.

3.4.2 Identification of candidate CK2 targets using peptide array target screens

Identification of a large number of candidate CK2/caspase targets that were phosphorylated by CK2 in the peptide target screens, which are implicated in the regulation of transcription and/or apoptosis, including Histone Deacetylase 4 (HDAC4) and Aven, supports a global role for CK2 phosphorylation directly in the regulation of cell survival. Previous studies investigating histone deacetylases as targets of CK2 showed that phosphorylation of HDAC1, HDAC2 and HDAC3 by CK2 was required for enzymatic activity, while phosphorylation of HDAC1 by CK2 has recently been shown to be a key activator of histone deacetylase activity in hypoxia-associated tumors, reiterating the essential role for CK2 in HDAC activity in cells [24-26]. The positive identification of phosphorylation of HDAC4 peptides by CK2 in peptide arrays at Ser565 (SDEEE) in conjunction with previously reported phosphorylation of HDAC 1, 2 and 3 suggests a role for CK2 in the regulation of HDAC4 deacetlyase activity. Alignment of the putative CK2 and caspase-8 and/or -9 consensus site within HDAC4 across species showed high conservation at these sites (Figure 3-8A). Interestingly, HDAC4 has been shown to cleaved by caspase-3 at Asp289, which results in the accumulation of the N-terminal fragment in the nucleus, facilitating the release of Cytochrome c and ultimately apoptosis [27]. The identification of a putative CK2/caspase site within HDAC4, which is a known caspase-3 target in cells, suggests a role for CK2 in the regulation of HDAC4 stability. However, further studies investigating the phosphorylation of HDAC4 in cells by CK2 at 95

Ser565 and its effect on caspase-3-mediated cleavage will be required. Notably, the location of the putative CK2 phosphorylation site within HDAC4 resides between two key residues modified by SUMOylation (Lys559) and phosphorylation (Ser632), which regulation deacetylase activity and nuclear export, further substantiating a role for CK2 phosphorylation in the HDAC4 deacetylatase activity [28, 29].

Aven, a protein involved in the negative regulation of apoptosis was identified in the CK2 peptide target screens to be phosphorylated at Ser94 (VEDDSDAE) adjacent to a putative caspase cleavage recognition site. Phosphorylation of Aven at Ser94 in cells was identified in large-scale phospho-proteomics studies; however, the protein kinase responsible had not been identified. The results of the current screens, provide evidence for CK2 as the kinase accountable for the phosphorylation of Aven at Ser94 [30]. Alignment of Aven sequences across species revealed conservation of the CK2 consensus site and the caspase recognition motif (Figure 3-8B). Previous studies have demonstrated a direct role for Aven in the protection of cells from apoptosis via the inhibition of the proteolytic activation of caspase-3 and -9 following stimulation of apoptosis [31].

Interaction between Aven and BAD (BC1-XL), a known CK2 target in cells, provides further evidence for a role for CK2 in the phosphorylation of Aven and the protection of cells from apoptosis [31, 32]. The mechanisms by which Aven is regulated in cells is only partial understood, however, one could envisage the requirement for the removal of Aven from the Apaf-l-caspase-9 signaling complex during the progression of apoptosis. Therefore, identification of a putative caspase cleavage site within Aven, a known associate of caspases, suggests a mechanism for the potential negative regulation of Aven via caspase-mediated cleavage. However, further studies to validate the CK2-dependent phosphorylation of Aven at Ser94 in cells, as well as an evaluation of the caspase sensitivity of Aven during the progression of apoptosis are required.

3.4.3 Identification of CHD8 as a promising overlapping CK2/caspase target using sPITS

Peptides corresponding to the transcriptional regulator CHD8 were phosphorylated by CK2 in peptide arrays as well as cleaved by caspase-3 in CSI assays, 96

B HDAC4 alignment AVEN alignment

Human EPIESDEEEAEP Human APVEDDSDAETYG Mouse EPIESEEEEAEA Mouse TRVEEDSDSETYG RAT EPIESDEEEAEP RAT TRVEEDSDSETYG Chicken EPIESDEEEAEP Cow EPIEDDSDAETYG D CHD8 alignment CHD4 alignment

Human LEDEDDSDSELDL Human DDLDVESDFDDAS Cow LEDEDDSDSELDL Cow DDLDVESDFDDAS Rat LEDDDDSDSELDL Rat DDLDVESDFDDAS Mouse LEDEDDSDSELDL Mouse DDLDVESDFDDAS Zebrafish IQGASDSDSD Zebrafish DDVDVDSDFDDGS

Figure 3-8 Alignment of predicted overlapping CK2 phosphorylation site and caspase cleavage motif in HDAC4, Aven, CHD4 and CHD8. The putative CK2 site phosphorylated in cells is highlighted in red, while the predicted caspase cleavage site is underlined. 97 suggesting a role for CK2 in the protection of CHD8 from caspase-mediated cleavage. Identification of phosphorylated peptides in large-scale mass spectrometry studies demonstrated that CHD8 was phosphorylated at Serl790 in cells, consistent with the phosphorylation of the Serl790 by CK2 in the peptide target screens [30]. A number of studies investigating the role of CHD8 in transcription showed that CHD8 was an essential participant in the regulation of a number of genes involved in cell survival. Specifically, through interactions with CCCTC-binding factor (CTCF), CHD8 was demonstrated to play a role in the epigenetic regulation of promoters within active insulator sites, including the BRCA1 and c-Myc genes [33]. Other studies investigating CHD8 as a transcriptional regulator showed that CHD8 suppressed the leukemia- inhibitory factor (LIF)-induced STAT3 transcriptional activity, suggesting a role for CHD8 in tumorigenesis [34, 35]. Recently, studies investigating the role of CHD8 in the regulation of gene transcription showed that CHD8 interacts directly with beta-catenin and negatively regulates beta-catenin-targeted gene expression. Interestingly, phosphorylation of beta-catenin by CK2 results in the stabilization and promotion of the beta-catenin-targeted genes, such as pro-survival signals [36, 37]. Downregulation of CHD8 has been shown to result in decreased histone acetylation and hypermethylation of BRCA1 and c-Myc promoter regions, as well as activation of several beta-catenin target genes [33, 38]. CK2 has been shown to bind to and phosphorylate both BRCA1 and c- Myc in cells, suggesting that CK2 may contribute in the regulation of the CHD8-targeted genes [39, 40]. Homologues of CK2 and CHD8 in yeast have been demonstrated to interact in a number of studies, supporting a functional relationship and a role for CK2 in chromatin remodeling [41]. However, further studies to determine whether CHD8 is cleaved by caspase-3 during apoptosis, as well as validation that CK2 phosphorylates CHD8 at Serl790 in cells are required. Notably, the putative Serl790 CK2 site in CHD8 was highly conserved across species, supporting a function for CK2 phosphorylation (Figure 3-8C). Previous studies using genome-wide expression screens in yeast implicated a global role for CK2 in chromatic remodeling which is consistent with a role for CK2 in the regulation of transcription via association with CHD8 [42]. Intriguingly, other family members of the Chromodomain Helicase DNA binding group, including CHD2, CHD4 and CHD7 were also identified in the bioinformatics search to have a CK2 98 consensus site for phosphorylation and/or caspase cleavage motif that were shown to be phosphorylated in cells, suggesting a role for CK2 in the global regulation of CHD transcription factors (Table 3-4). A species-conserved overlapping CK2/caspase target site at Ser319 was also identified in CHD4 providing further evidence for a role for CK2 in the regulation of CHD proteins (Figure 3-8D). It will be of interest to explore further the molecular mechanisms involved in the regulation of CHD-regulated genes and to determine whether CK2 functions positively or negatively in the modulation of chromatin structure associated with CHD activity.

3.4.4 Phosphorylation of the P2 or PI' within proteolytic activation cleavage site of caspase-3 prevents cleavage

The progression of apoptosis requires the cleavage of inactive pro-caspases into active proteases resulting in the cleavage of a large number of proteins required for cell survival including PARP, Fodrin, as well as key signaling proteins such as PTEN and Max [43]. Identification of pro-caspase-3 and pro-caspase-8 from the bioinformatics search as candidate substrates for CK2 phosphorylation and caspase cleavage, implicate a potential role for CK2 in the direct regulation of caspase-signaling pathways. Generation of pro-caspase-3 into its active form requires caspase-8 or -9 processing at Aspl75, resulting in the downstream cleavage of caspase-3 targets and progression of apoptosis [1, 44]. In the present study, the effect of phosphorylation on caspase-mediated cleavage of caspase-3 by caspase-8 was investigated via the synthesis of peptides that were phosphorylated at the P2 and PI' residues adjacent to Asp 175 cleavage site. Phosphorylation of the P2 or PI' residues within the caspase-3 peptides was shown to prevent caspase cleavage in the in vitro cleavage assay demonstrating that phosphorylation within caspase-3 activation cleavage site would presumably result in the prevention of the maturation of pro-caspase-3 into active caspase-3. Regulation of pro- caspase-3 and -8 maturation into active forms by CK2 phosphorylation could provide a potential mechanism where CK2 functions directly in apoptosis via the control of caspase activation. The participation of CK2 in the initiation and execution stages of apoptosis could represent a novel mechanism for prevention of apoptosis; however, further studies 99

Table 3-4. Members of the Chromodomain Helicase DNA binding protein (CHD) family phosphorylated at a CK2 consensus sites in cells. Phosphorylated site highlighted in red.

Swiss-Prot ID Sequence Phospho-Site in Cells KRQIDSSEEDDDEEDY Ser215 CHD1 HUMAN KRQIDSSEEDDDEEDYD Ser216 KRKKRDSEEEFGSERDE Ser73 CHD3 HUMAN SEEEFGSERDEYREKSE Ser79 DPEEDLSETETPKLKKK Ser44 GSKRKRSSSEDDDLDVE Ser 308 SKRKRSSSEDDDLDVES Ser 309 CHD4 HUMAN KRKRSSSEDDDLDVESD Ser310 DDLDVESDFDDASINSY Ser319* EAKEDNSEGEEILEEVG Ser 428 QAPAPASEDEKVVVEPP Ser1602 DDDLVEFSDLESEDD Ser1141 CHD8 HUMAN KLEDEDDSDSELDLS Ser1790* EDEDDSDSELDLSKL Ser1792 KQKRKNESSDEISDA Ser 611 CHD9 HUMAN ESSDEISDAEQMPQ Ser 616 KDELAELSEAESEGD Ser1468 Peptides phosphorylated in CK2 peptide target screens 100 to determine whether pro-caspase-3 and/or pro-caspase-8 are phosphorylated by CK2 in cells, as well as the effect of phosphorylation on caspase activation are required.

In summary, a sequence-based protein identification targeting strategy (sPITS) was utilized to investigate the global role of phosphorylation in the regulation of caspase signaling. A comprehensive evaluation of overlapping protein kinase CK2 and caspase targets revealed over 300 proteins with a CK2 phosphorylation consensus site at the P2 or PI' position within a caspase recognition sequence. A number of peptides corresponding to proteins containing an overlapping CK2/caspase consensus were shown to be phosphorylated in the CK2 peptide array target screens, as well as cleaved by caspases using CSI, representing novel candidate CK2/caspase targets. Demonstration that pro- caspase-3 peptides were protected from caspase-mediated cleavage by phosphorylation at the P2 and/or PI', suggests a role for phosphorylation in the proteolytic maturation of pro-caspase-3. Evidence that phosphorylation at the P2 or PI' residues within the caspase cleavage recognition motif is sufficient to prevent caspase cleavage, as well as the identification of numerous candidate CK2/caspase targets in the present studies, support a role for protein kinases in the regulation of caspase signaling. 3.5 References

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[38] Thompson, B. A., Tremblay, V., Lin, G. and Bochar, D. A. CHD8 is an ATP- dependent Chromatin Remodeling Factor That Regulates {beta}-catenin Target Genes, Mol Cell Biol 28,3894-904.

[39] Channavajhala, P. L. and Seldin, D. C. (2002) Functional interaction of protein kinase CK2 and c-Myc in lymphogenesis, Oncogene 21,5280-5288.

[40] O'Brien, S., Lemke, S. J., Cocke, K. S., Rao, R. N. and Bechmann, R. P. (1999) Casein kinase 2 binds to and phosphorylates BRCA1, Biochem Biophys. Res. Commun. 260,658-664.

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[42] Barz, T., Ackermann, K., Dubois, G., Eils, R. and Pyerin, W. (2003) Genome-wide experession screens indicate a global role for protein kinase CK2 in chromatin remodeling, J Cell Sci 116,1563-1577.

[43] Fischer, U., Janicke, R. U. and Schulze-Osthoff, K. (2003) Many cuts to ruin: a comprehensive update of caspase substrates, Cell Death Differ 10,76-100.

[44] Slee, E. A., Adrain, C. and Martin, S. J. (2001) Executioner Caspase-3, -6, and -7 Perform Distinct, Non-redundant Roles during the Demolition Phase of Apoptosis, J. Biol. Chem. 276,7320-7326. 106

CHAPTER 4 REGULATION OF PRO-CASPASE-3 PROTEOLYTIC ACTIVATION BY CK2: A MECHANISM FOR PROTECTION OF CELLS FROM APOPTOSIS 4.1 Introduction

Apoptosis is an essential biological process required for normal cell turnover, proper embryonic development, as well as chemically induced cell death. However, perturbations in the regulation of apoptosis signaling pathways has been associated with many human diseases, including cancer [1]. The loss of regulation of key protein kinases within these apoptotic pathways has been demonstrated to contribute to the protection of cells from apoptosis during tumorigenesis [2, 3]. CK2 (formally known as, Casein Kinase II) is a constitutively active, ubiquitously expressed serine/threonine protein kinase that has been implicated in the regulation of multiple cellular processes including cell survival, apoptosis and tumorigenesis [4]. CK2 has been shown to promote tumorigenesis via the regulation of key tumor suppressors and oncogenes within pro- survival signaling pathways [4, 5]. Consequently, an understanding of the molecular mechanism by which CK2 functions in tumorigenesis has been an area of intense research, with an emphasis on targeting CK2 for anti-cancer therapeutics.

An anti-apoptotic role for CK2 has emerged following studies indicating that inhibition of CK2 in various cancer cells sensitized cells to receptor-mediated and intracellular apoptosis [6, 7]. An essential role for CK2 in apoptosis was illustrated in prostate cancer cells in which overexpression of CK2a resulted in the protection of cells from TRAIL/Apo2L-mediated apoptosis [8]. Furthermore, treatment with CK2 inhibitors was shown to sensitize cells to TRAIL-induced apoptosis, resulting in increased formation of the death inducing signaling complex (DISC), enhanced caspase 8 and Bid cleavage, as well as down-regulation of the inhibitors of apoptosis (IAP) proteins, xIAP and c-IAP [9]. Similar studies using RNA interference to knockdown CK2 also resulted in sensitization of cells to apoptosis following 6-TG-induced DNA damage, demonstrating that CK2 was required for inhibiting apoptosis, as well as functioning in the control of caspase activity [10]. Interestingly, elevated CK2 levels in cells has been linked to the regulation of expression of a key IAP protein, survivin, which inactivates 107 caspases and stabilizes other IAPs, promoting cell survival and tumorigenesis [11]. Increasing evidence suggests that CK2 functions within apoptotic signaling pathways and that alterations in CK2 levels in cells can influence the apoptotic state of the cell.

Sensitization of cancer cells to apoptosis via the interference of CK2 activity has prompted multiple studies to address the molecular mechanism by which CK2 functions in the protection of cells from apoptosis. Promotion and repression of the activity of key proteins within apoptosis signaling pathways by CK2 has been demonstrated supporting an anti-apoptotic role for CK2 [2]. Phosphorylation of pro-caspase-2 by CK2 has been shown to prevent dimerization and activation, while phosphorylation of the caspase inhibiting protein, ARC, facilitates the inhibition of caspase-8 activity, both of which prevent the progression of apoptosis [12, 13]. Intriguingly, phosphorylation of a number of pro-survival proteins by CK2 at or near the caspase cleavage site resulted in protection from caspase mediated cleavage, thereby promoting cell survival (Table 4-1). An overlapping requirement for acidic residues in the CK2 phosphorylation consensus motif and the caspase recognition cleavage sequence suggests a global role for CK2 in the regulation of a wide variety of caspase targets within the apoptotic signaling pathways [4]. The loss of regulation of apoptosis has been associated with most cancers, characterized by the near complete absence of caspase activation, reiterating the importance of understanding how CK2 exhibits its anti-apoptotic function within caspase signaling pathways.

In the present study, an evaluation of the molecular mechanism by which CK2 functions within caspase signaling pathways was undertaken. A proteome-wide bioinformatics search for proteins with overlapping CK2 phosphorylation motif and caspase cleavage recognition sequence identified pro-caspase-3 as a putative target. Demonstration that CK2 phosphorylation can protect pro-caspase-3 from caspase mediated cleavage and that loss of CK2 activity results in an increase activation of caspase-3 provides a novel molecular mechanism by which CK2 protects cells from apoptosis. 108

Table 4-1. Proteins protected from caspase-mediated cleavage by CK2 phosphorylation. CK2 phosphorylated residues highlighted in red, while caspase cleavage sites underlined. References are illustrated in brackets. Overlapping CK2 and Protein Name Caspase caspase cleavage site

BID [18] DELQTDGSQ 8 Presenilin-2 [32] DSYDSFGEP 3 Max [33] IEVESDEEQP 5 PTEN [34] DVSDNEPDH 3 PTEN [34-36] DTTDSDPEN 3 Connexin45.6[37] VVSDEVEGP 3 109

4.2 Materials and Methods

4.2.1 Cell culture and transfections

HeLa cells were cultured in DMSO (Gibco) containing 10% fetal bovine serum (Gibco), penicillin (100 U/mL) and streptomyocin (100 fxg/mL) (Gibco) on 10 cm plates (Falcon). HeLa cells were transfected with either CK2o>HA/HA-CK2p\ CK2a-HA (K68M)/HA-CK2p in addition to pro-caspase-3-myc (Addgene) or CK2cx-shRNA, CK2cx'-shRNA, scramble-shRNA using calcium phosphate with a transfection efficiency of > 80%. For CK2 inhibition studies, cells were treated with 4,5,6,7-Tetrabromo-lH- benzotriazole (TBB) (Calbiochem) or etoposide (Calbiochem).

4.2.2 Generation ofpro-caspase-3 mutants and purification

Catalytically inactive His-tagged pro-caspase-3 (ATCC) was engineered using a QuickChange II Site-Directed Mutagenesis Kit (Stratagene). Human pro-caspase-3 pET23b was PCR amplified to make the (CI63A) mutation using the primer 5- TTCATTATTCAGGCCGCCCGTGGTACAGAA-3'. Phosphorylation mutants (T174A), (SI76A) and (T174A SI76A) of pro-caspase-3 were generated using the following primers 5-GACTGTGGCATTGAGGCAGACAGTGGTGGTGAT-3', 5"

GGCATTGAGACAGACGCTGGTGGTGATGAC-3', 5-

GACTGTGGCATTGAGGCAGACGCTGGTGTTGATGATGAC-3', respectively. Pro-caspase-3 (CI63A) and the phosphorylation mutants were purified with a HiTrap SP HP column (Amersham Biosciences) using the AKTA Purifier FPLC (Amersham Biosciences).

4.2.3 Knockdown and inhibition studies

HeLa cells were co-transfected with CK2a-shRNA targeting sequence (5 - TGGGGTAATCAAGATGATT-3'), CK2a'-shRNA targeting sequence (5>- GAACCTTCGTGGTGGAACA-3') or scramble-shRNA targeting a random sequence (5-

ACTGTACATCGCACTTCTG-3) along with truncated CD4. Cells were magnetically cell sorted 72 hrs post transfection to enrich the transfected population (MACSelect Transfect Cell Selection Kit, Miltenyi Biotech). HeLa cells were lysed and Western blot analysis was carried out to visualize CK2 knockdown and to investigate apoptosis by assessing 110 cleaved PARP levels in each treatment. Chemical inhibition of CK2 was investigated following treatment of HeLa cells for 6, 12 and 24 hrs with TBB at a concentration of 25 \iM. Following inhibitor treatment cells were lysed and Western blot analysis was performed. HeLa cells co-transfected with CK2ct-HA/HA-CK2p and/or pro-caspase-3- myc were also treated with 25 uM TBB for 12 hrs, following which the cells were lysed and subjected to Western blot analysis.

4.2.4 Immunoblot analysis

HeLa cells were harvested from 10 cm plates in NP40 lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mg/mL Leupeptin, 1 mM PMSF, 1 mg/ml Pepstatin, 5 ug/ml Aprotinin, 1 ^iM Okadaic Acid and 1/100 of phosphatase inhibitor set II (Calbiochem). Lysates were sonicated and protein concentrations determined by BCA assay. Proteins were resolved on 10% and 12% SDS-PAGE electrophoresis followed by transfer to PVDF (Millipore). Membranes were blocked with LI-COR blocking agent (LI-COR Biosciences) or 5% BSA/TBST for 1 hr followed by overnight incubation at 4 °C with the primary antibody in 50% PBS/Licor blocking agent or 1% BSA/TBST. Membranes were probed with primary antibodies including anti-PARP (Cell Signaling), anti-Caspase-3 (Cell Signaling), anti-P-tubulin (Sigma), and/or anti-c-myc 9E10 biotin (Berkeley Antibody Company). Membranes were washed with TBST then incubated with secondary antibodies, (LI-COR Biosciences) 680-GAR, 800-GAR 680-GAM, 800-GAM, 680-SAV, 800-SAV or HRP-GAR (BioRAD), and/or HRP-biotin (Jackson ImmunoResearch). Following incubation the membranes were washed with TBST and visualized via LI-COR Odyssey Fluorescent imager (LI-COR Biosciences) or by enhanced chemiluminescence (ECL) (Amersham Pharmacia). The X- ray film (Kodak) was developed and converted to a digital image using CanoScan N650U/N656U scanner at 600 Dpi. Images were visualized via Adobe Photoshop CS.

4.2.5 Immunoprecipitations

HeLa cells co-transfected with CK2a-HA/HA-CK2|3 and pro-caspase-3-myc were grown on 10 cm plates (Falcon), harvested using PBS-EDTA and lysed in NP40 lysis buffer as described previously. Following determination of protein concentration by Ill

BCA assay (Pierce), equal amounts of protein were incubated with 50 uL of Protein G sepharose (Amersham Pharmacia) or Protein-A-sepharose (Amersham Pharmacia) beads and 1-5 u.g of anti-myc 9E10 (Berkeley Antibody Company) or 12CA5 HA (Roche) antibodies, respectively. Following an overnight incubation at 4°C, beads were isolated by centrifugation and washed extensively. Beads were then incubated in SDS loading buffer, boiled for 5 min and centrifuged briefly to remove beads. Proteins were resolved on 10% and 12% SDS-PAGE electrophoresis, transferred to PVDF membranes followed by Western blot analysis. Membranes were probed with anti-HA biotin 3F10, anti-c-Myc 9E10, and/or anti-caspase-3 antibodies.

4.2.6 Phosphorylation ofcaspase-3 by CK2 in vitro

In vitro kinase assays were performed using recombinant His-tagged full-length inactive pro-caspase-3 (CI 63 A). For phosphorylation, 2 u.g of purified pro-caspase-3, the single phosphorylation site mutants pro-caspase-3 (S176A), pro-caspase-3 (T174A) or double phosphorylation site mutant (T174A S176A) were incubated in 50 mM Tris-HCl 32 pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.1 mM ATP, 0.2 uCi [y- P]-ATP (Perkin Elmer) and 2 U of recombinant GST-CK2a for 20 min at 30 °C. Reactions were stopped by the addition of SDS sample buffer and proteins were resolved by 10% SDS-PAGE gel electrophoresis. Gels were then dried and radiolabeled proteins were visualized using a Phosphorimager (Molecular Dynamics). The relative amount of [y-32P]-ATP incorporated was determined using ImageQuant TL software (Amersham Biosciences).

4.2.7 32P cell labeling and TBB treatment

HeLa cells co-transfected with CK2a-HA/HA-CK2|3 and caspase-3-myc were labeled with carrier-free [y-32P] orthophosphate (Perkin Elmer) by washing cells extensively with DMEM (PO4-) specialty media (Chemicon Specialty Media's). The cells were then cultured in phosphate-free medium supplemented with 1.67 mCi [y-32P] orthophosphate and either 60 uM TBB or DMSO for 4 hrs [14, 15]. Following in vivo 32P label, cells were harvested and pro-caspase-3-my c was immunopreciptated as described earlier and radiolabeled proteins were resolved on a 10% SDS-PAGE electrophoresis, dried and visualized on the phosphorimager with analysis using 112

ImageQuant TL software. The relative amount of [y- P]-ATP incorporated was determined using ImageQuant TL software (Amersham Biosciences).

4.2.8 Cleavage of pro-caspase-3 by caspase-8 and -9.

To test the effect of phosphorylation of pro-caspase-3 on caspase-mediated cleavage, 2 [xg of purified pro-caspase-3 was incubated with GST-CK2a in a kinase assay as outlined previously. Following the kinase assay, 6 U/ul of caspase-8 (BIOMOL) or caspase-9 (BIOMOL) was added to the kinase reaction in caspase assay buffer containing (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM DTT) for caspase-8 or (100 mM MES, pH 6.5, 10% PEG (Polyethylene Glycol, average MW 8000), 0.1% CHAPS, 10 mM DTT) for caspase-9 reactions. Cleavage reactions were incubated overnight at 30 °C with gentle agitation to obtain optimal cleavage. Cleavage reactions were stopped by the addition of SDS sample buffer. Radiolabeled proteins were resolved on 12% SDS-PAGE electrophoresis, and the gels were dried and visualized on the phosphorimager. Determination of the amount of [y-32P]- ATP incorporated was assessed using ImageQuant TL software. Non-radiolabeled cleavage reactions were performed in parallel to verify caspase-8 and -9 activity. Following a "cold" kinase assay (no [y-32P]-ATP) incubating 2 U of recombinant GST- CK2aand 2 u.g pro-caspase-3, pro-caspase-3 (S176A), or (T174A/S176A) in 50 mM

Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.1 mM ATP for 20 min at 30 °C, an in vitro caspase cleavage assay using 6 U/ul of caspase-9 was performed, as outlined previously. Cleavage assays were stopped by the addition of SDS sample buffer and proteins were resolved on 12% SDS-PAGE electrophoresis, transferred to PVDF and probed with anti-caspase-3 antibodies or stained with Gel Code Blue (Pierce). All reactions were performed in triplicate. The percentage of cleaved caspase-3 (17 kDa) compared to full length pro-caspase-3 (32 kDa) was determined and a statistical analysis was performed using ANOVA. 113

4.2.9 Assay ofcaspase-3 activity in cell lysates

Caspase-3 activity was assessed using the Ac-DEVD-pNA (P412) colorimetric substrate (BIOMOL). HeLa cells transfected with CK2a-(K68M)-HA/CK2(3 or CK2o> HA/CK2P and pro-caspase-3-myc were treated with DMSO or 25 uM TBB for 12 hrs and harvested in NP40 lysis buffer. Twenty micrograms of cell lysate was incubated in caspase-3-assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 mM DTT) with 200 uM Ac-DEVD-pNA substrate in 96 well plates (Evergreen Scientific) and continuously monitored at A^snm via the Victor 3V system (Perkin Elmer). All treatments were performed in triplicate. The linear portion of the Abs vs. time graph was used to determine the rate of caspase-3 cleavage, as well as the units of activity of caspase-3 in the differently treated cell lysates. Caspase-3 activity (pmol/min/fxg) was calculated for each treatment and a statistical analysis comparing the treatments was performed using ANOVA.

4.3 Results

4.3.1 Interference with CK2 activity results in spontaneous apoptosis

Chemical inhibition of CK2 or downregulation of CK2 protein levels has been previously shown to sensitize cells to apoptosis following treatment with an apoptotic stimulus, reiterating the emerging anti-apoptotic role for CK2 in regulation of cell survival [6, 7]. To further characterize the role of CK2 in apoptosis, and to test the involvement of CK2 in caspase signaling, an evaluation of the effect of chemical inhibition of CK2 on HeLa cell survival in the absence of a death stimulus was performed using the specific CK2 inhibitor, TBB (Figure 4-1 A). HeLa cells were treated with either DMSO, 25 \xM TBB or 25 uM etoposide for 6, 12 and 24 hrs. The cleavage of PARP, a known target of caspase-3, was used as an indicator of apoptosis and revealed that treatment of cells with TBB resulted in the generation of a cleaved PARP (89 kDa) band following 24 hrs of treatment as compared to the DMSO control. Etoposide treatment was used as a positive control inducing apoptosis in HeLa cells following 24 hrs of treatment. To explore this further, downregulation of CK2a and CK2a' was performed using RNA interference (Figure 4-IB). HeLa cells transfected with CK2a- 114

•v ,§•

p-tubulin-

B CK2a •

CK2a' •

p-tubulin •

CK2a-shRNA CK2a'-shRNA Scram-shRNA

PARP -J Cleaved PARP "•

p-tubulin —I

CK2

Figure 4-1. Spontaneous apoptosis is associated with interference with CK2 function. (A) HeLa cells were treated with DMSO or 25 uM TBB for 6, 12 and 24 hrs to investigate the role of CK2 in caspase signaling and progression of apoptosis. Cells were harvested and lysates were examined for the presence of an 89 kDa cleaved PARP band as an indicator of apoptosis using Western blot analysis via probing membranes with anti- PARP antibodies. (B) Levels of the 45 kDa CK2a band as well as the 38 kDa CK2a' band following treatment with RNA interference targeting the CK2 catalytic subunits in HeLa cells was evaluated by Western blot analysis using anti-CK2a and anti-CK2a' antibodies respectively. A scramble-shRNA sequence was used as a control for off-target effects. (C) Generation of the 89 kDa cleaved PARP band was assayed as an indicator of apoptosis in HeLa cells treated with RNA interference against the catalytic subunits CK2 via Western blot analysis probing membranes with anti-PARP antibodies. Blots were probed with anti-|3-tubulin antibodies to assure equal loading across treatments and etoposide was used as a positive control for apoptosis. 115 shRNA or CK2oc'-shRNA reduced CK2 levels in cells and induced apoptosis as evident by the generation of a cleaved PARP (89 kDa) band as compared to the control scramble- shRNA treated and untreated cells (Figure 4-1C). Therefore, it appears that interference with CK2 activity in HeLa cells results in spontaneous apoptosis independent of a death stimulus, implicating a direct link between CK2 and the progression of caspase dependent apoptosis.

4.3.2 Phosphorylation of pro-caspase-3 by CK2 protects from cleavage by caspase-8 and-9

A number of CK2 targets have been identified that are essential constituents in the apoptotic response, where phosphorylation by CK2 resulted in protection from caspase mediated cleavage [5]. The overlapping requirement of an acidic consensus motif for CK2 phosphorylation recognition (S/T-X-X-D/E) and the aspartic residue required for caspase cleavage prompted a proteome-wide bioinformatics evaluation of CK2 and caspase overlapping targets [1, 16]. Intriguingly, many candidate proteins involved in apoptosis signaling were identified, including pro-caspase-3. Caspase-3 has been shown to be one of the key effector caspases in which both the intrinsic and external apoptotic signals converge resulting in the activation of caspase-3 and the subsequent proteolytic cleavage of multiple targets during apoptosis [17]. Notably, the putative CK2 site that was identified, Serl76, falls within the activation cleavage site at the PI' position adjacent to Aspl75, which is cleaved by caspase-8 and-9 during apoptosis [1]. Alignment of pro-caspase-3 across species revealed that the CK2 site is conserved (Figure 4-2A) suggesting that Serl76 could be important in the regulation of the activation of caspase-3. To test whether pro-caspase-3 was an in vitro target of CK2, a kinase assay was performed with GST-CK2a and purified catalytically inactive pro-caspase-3 (C163A). Identification of a 32 kDa pro-caspase-3 radiolabeled band revealed that pro- caspase-3 was indeed phosphorylated by CK2. To validate that the phosphorylation of caspase-3 was by GST-CK2cc and not an artifact of protein purification, the CK2 inhibitor TBB was employed, resulting in the expected reduction of the incorporation of [y-32P] into caspase-3. 116

M_P1' w B Human GIETDSGVDD 180 Rat GIETDSGTDD 180 MOUM GIITDSGTDE 180 [»Mp] Dog GXETDSGIKD 180 PiO GIETOSGTED 180 pro-caspase-3 Flah GIETOSG-ED 184 (32 kDa) -»

ppj

pro-caspase-3 (32 kDa) -• GST-CK2a pro-caspase-3 caspase-8 pro-caspase-3 caspase-9 GST-CK2u TBB

IB: caspase 3 IB: caspase 3 ••.!• 11^ in) ••pimp pro-caspase-3 pro-caspase-3 (32 kDa) -• (32 kDa) -H

Cleaved fc caspase-3 (17 kDa) (17 kDa) "

GST-CK2a GST-CK2a pro-caspase-3 pro-caspase-3 caspase-8 caspase-9 caspase-9 ATP

Figure 4-2. Phosphorylation of pro-caspase-3 by CK2 results in protection from caspase mediated cleavage. (A) An alignment of the pro-caspase-3-protein sequence from different species revealed a conserved CK2 phosphorylation motif (Ser 176) within the caspase activation sequence of pro-caspase-3 at the PI' position adjacent to Asp 175. In vitro [y-32P] kinase assays using GST-CK2a and purified pro-caspase-3 (CI63A) as a substrate were performed to determine whether pro-caspase-3 was an in vitro target of CK2. Treatment of kinase reactions with 60 uM TBB was used as a control. (B) An in vitro [y-32P] kinase assay with GST-CK2a and pro-caspase-3 was performed followed by treatment with active caspase-8 or -9. Generation of [y- P] labeled caspase-3 cleavage products was then analyzed to investigate the phosphorylation dependent protection of pro-caspase-3 from caspase-mediated cleavage by CK2. (C) In parallel, an in vitro "cold" kinase assay of GST-CK2a and pro-caspase-3 followed by treatment with active caspase- 8 or -9 was also performed. The generation of a (17 kDa) cleaved caspase-3 fragment following treatment of pro-caspase-3 with active caspase-8 and -9 was assayed by Western blot analysis using anti-caspase-3 antibodies to validate that caspase-8 and -9 were active in the in vitro caspase cleavage assays. (D) An in vitro "cold" kinase assay of GST-CK2a and pro-caspase-3 in the presence and absence of ATP was performed to test the requirement for CK2 activity in the protection from caspase-mediated cleavage. Cleaved (17 kDa) pro-caspase-3 was determined via Western blot analysis using anti- caspase-3 antibodies. 117

The protection from caspase-mediated cleavage by CK2 phosphorylation has been documented in a number of key apoptosis regulating proteins. For example, phosphorylation of the pro-apoptotic protein Bid by CK2 was shown to prevent its cleavage by caspase-8, thereby inhibiting the progression of Fas-mediated apoptosis [18]. The discovery of pro-caspase-3 as a putative CK2 target further reinforces the prospect of a role for CK2 in the protection of cells from apoptosis. To test whether phosphorylation of pro-caspase-3 by CK2 protected pro-caspase-3 from caspase-8 and -9 mediated cleavage, in vitro caspase cleavage assays were performed. Firstly, in vitro kinase assays were performed with GST-CK2cc and pro-caspase-3, followed by incubation with active caspase-8 and-9 to investigate whether the [y-32P]-labeled pro-caspase-3 could be cleaved (Figure 4-2B). A single 32 kDa radiolabeled pro-caspase-3 band with no 17 kDa [y-32P] cleavage product was observed in the CK2 and caspase-8 and-9 treatments indicating that phosphorylation of pro-caspase-3 by CK2 resulted in the protection from caspase-8 and-9 mediated cleavage. In parallel, a "cold" kinase assay was performed followed by a caspase-8 and-9 cleavage assay to validate that the caspases were functional. The cleavage assays revealed that caspase-8 and -9 were indeed cleaving pro-caspase-3 as evident by the generation of cleaved (17 kDa) caspase-3 products on the Western blot probed with anti-caspase-3 antibodies (Figure 4-2C). The protection of pro-caspase-3 by CK2 phosphorylation was also observed by Western blot analysis as indicated by the reduced amount of cleaved (17 kDa) caspase-3 band in the CK2 treatments compared to the cleavage of caspase-3 by caspase-8 and -9 in the absence of CK2. Finally, to test whether the activity of CK2 was essential for the protection of pro-caspase-3 from cleavage, the cleavage of pro-caspase-3 by caspase-9 was tested in the presence and absence of ATP (Figure 4-2D). These assays demonstrated that active CK2 was critical for the observed protection as evident by an increase in (17 kDa) cleaved caspase-3 band in the absence of ATP. Collectively, these experiments indicate that phosphorylation of pro-caspase-3 by CK2 was found to protect it from caspase-8 and -9 cleavage. However, further studies to identify the phosphorylation sites involved in the protection, and to test whether the predicted Serl76 putative site was phosphorylated, are required. 118

4.3.3 Phosphorylation of Thrl74 and Serl76 by CK2 protect pro-caspase-3 from caspase-mediated cleavage

Phosphorylation of a Ser residue at the PI' site adjacent to the caspase cleavage site has been demonstrated to abolish hydrolysis of caspase target peptides, indicating that the addition of a large phosphate sterically hinders the ability of the aspartic protease to cleave at the adjacent residue [19]. Intriguingly, the putative Serl76 CK2 site within pro- caspase-3 falls at the PI' site adjacent to Asp 175 suggesting that if the site were indeed phosphorylated it would presumably interfere with caspase-8 and -9 mediated cleavage. To test whether Serl76 was phosphorylated and involved in the protection of pro- caspase-3 from cleavage by caspase-8 and-9, a mutant form of pro-caspase-3 was engineered with Ser 176 replaced by an Ala (S176A). In vitro kinase assays were performed using the SI76A mutant and GST-CK2a, which showed a reduction in [y-32P] incorporation, as evident by a decrease in [y-32P] 32 kDa pro-caspase-3 band. The incomplete reduction in [y- P] incorporation with the SI76A mutants compared to pro- caspase-3 demonstrated that Ser 176 was indeed phosphorylated by CK2; however, it was not the sole CK2 phosphorylation site in pro-caspase-3 (Figure 4-3 A). Further analysis of the pro-caspase-3 protein sequence, revealed another potential CK2 site, Thrl74 at the P2 position adjacent to Aspl75 (Figure 4-2A). To test whether Thrl74 was phosphorylated by CK2, mutant forms of pro-caspase-3 were generated, where Thrl74 was mutated to Ala (T174A) as well as the generation of a double site mutant (T174A/S176A). CK2 kinase assays were performed with the mutant forms of pro-caspase-3 and revealed that Thrl74 was also phosphorylated by CK2 indicated by a reduction in [y-32P] incorporation and that when both (T174A/S176A) were mutated phosphorylation by CK2 was completely abolished, as evident by the loss the [y-32P] (32 kDa) pro-caspase-3 band. Overall, these experiments mapped the CK2 phosphorylation sites within pro-caspase-3 to Thrl74 and Ser 176, however, elucidation of the function of phosphorylation of Ser 176 and Thrl74 by CK2 in the protection of pro-caspase-3 from cleavage required further evaluation.

To characterize the protective effect of phosphorylation of pro-caspase-3 by CK2 at Thrl74 and Ser 176 studies investigating the protection of CK2 phosphorylation on 119

B C3 S176A T174A T174A IB:caspase 3 CK2 CK2 CK2 S176A

M CK2 [» P] pro-caspase 3 pro-caspase-3 3, (32 kDa) *" (32 kDa) *•'

c 2§ o [T»p] cleaved (17 kDa) 8. o 60 • §sS : pro-caspase-3 + : 1- BBB T174AS176A + 9- *8 - * I I ™" I t Caspase-9 + + C3 SA TA TSA GST-CK2a + + TBB +

Stain: Gel code blue pro-caspase 3 (32 kDa) *"

C9 cleaved >. (17 kDa) Pro-caspase-3 T174A/S176A Caspase-9 C3 T174A S176A GST-GK2a

Figure 4-3. Phosphorylation of pro-caspase-3 at Thrl74 and Serl76 protects from caspase-mediated cleavage. (A) In vitro kinase assays using pro-caspase-3, pro-caspase-3 (S176A), pro-caspase-3 (T174A) or pro-caspase-3 (T174A/S176A) mutants and GST- CK2ct (CK2) were performed to map the CK2 phosphorylation sites in pro-caspase-3. [y-32P] incorporation into pro-caspase-3 for each treatment was calculated based on the relative [y-32P] incorporation determined by ImageQuant TL software. (B) In vitro caspase-9 cleavage assays of pro-caspase-3 and the double phosphorylation site mutant (T174A/S176A) were performed following incubation with GST-CK2a. Western blot analysis was carried out probing membranes with anti-caspase-3 antibodies for cleaved (17 kDa) pro-caspase-3. Treatment of kinase reactions with 60 uM TBB or in the absence of GST-CK2a were used as controls. (C) Cleavage of pro-caspase-3 and the (T174A/S176A) mutant was visualized on Gel Code Blue stained gels following CK2 and/or caspase-9 treatment. ANOVA analysis (P< 0.01) was performed to compare the percentage of cleaved 17 kDa band to full-length 32 kDa pro-caspase-3 band between pro-caspase-3 and the pro-caspase-3 (T174A/S176A) mutant. Error bars represent standard deviation within treatments. 120 caspase cleavage were then undertaken using the pro-caspase-3 phosphorylation site (T174A/S176A) mutants. In vitro caspase cleavage assays were performed, which demonstrated an increase in the cleaved caspase-3 (17 kDa) band with the (T174A/S176A) mutants following caspase-9 treatment compared to that of caspase-3 in Western blots using an anti-caspase-3 antibody (Figure 4-3B). The phosphorylation site mutants were cleaved by caspase-9 comparable to that of conditions where CK2 was inhibited using TBB and approaching that of caspase-9 treatment alone. A statistical difference in the percentage of pro-caspase-3 and (T174A/S176A) mutants cleaved by caspase-9 was determined via comparing the amounts of cleaved caspase-3 (17 kDa fragment) to full length pro-caspase-3 on Gel Code Blue stained gels, indicating that mutation of the CK2 phosphorylation sites Thrl74 and Serl76 resulted in the loss of protection from caspase cleavage (Figure 4-3 C).

4.3.4 Interaction of CK2 and pro-caspase-3 in cells; evidence for CK2 dependent phosphorylation of pro-caspase-3

Phosphorylation of pro-caspase-3 at Thrl74 and Serl76 within the activation cleavage site resulted in the protection from caspase-8 and -9 mediated cleavage in vitro. Accordingly, the regulation of activation of caspase-3 by phosphorylation could provide a mechanism by which CK2 may protect cells from apoptosis as well as facilitate tumorigenesis. A number of post-translational modifications of pro-caspase-3, including nitrosylation, ubiquitination and glutathiolation have been implicated to play a role in maturation of active caspase-3; however, the exact mechanism by which the different modifications contribute in the activation of caspase-3 remains unknown [20-22]. In the current study, identification of phosphorylation of pro-caspase at the activation cleavage site by CK2 presents another potential post-translational modification required for protection of apoptosis via the prevention of caspase-3 activation. To investigate whether pro-caspase-3 and CK2 exist in complex, co-immunoprecipitation experiments were performed. Immunoprecipitation of CK2a-HA and HA-CK2P revealed a 32 kDa pro- caspase-3 band in Western blots probed with anti-caspase-3 antibodies (Figure 4-4A). In parallel, CK2a-HA and HA-CK2|3 were identified in immunoprecipitation of pro- caspase-3-myc as indicated by the 50 kDa CK2a-HA and 30 kDa HA-CK2|3 on Western 121

CK2a-HA|

IB: anti-HA HA-CK20*

IB: anti-caspase-3 pro- caspase-3-*- myc 3: B [yMpJ

IP: Myc [V32P]

IP: Myc IB: pro-caspase-3

pro-caspase-3 CK2a-HA TBB

Figure 4-4. CK2 interacts with and phosphorylates pro-caspase-3 in cells. (A) Immunoprecipitation of pro-caspase-3-myc was performed with anti-myc 9E10 antibodies from HeLa cells co-transfected with CK2a-HA, HA-CK2|3 and pro-caspase-3- myc. Following immunoprecipitation of pro-caspase-3-myc, Western blot analysis probing membranes with anti-HA 3F10-biotin conjugated antibodies was carried out. In parallel, immunoprecipitation of CK2o>HA and HA-CK2|3 was performed using anti-HA 12CA5 antibodies, followed by Western blot analysis probing membranes with anti- caspase-3 antibodies. Control precipitations were performed without 9E10 antibodies. (B) [y-32P] labeled HeLa cells co-transfected with CK2a/|3 and pro-caspase-3-myc were treated with 60 uM TBB or DMSO for 4 hrs, followed by anti-myc immunoprecipitation of pro-caspase-3-myc using anti-myc 9E10. Relative [y-32P] incorporation was determined using ImageQuant TL software. In parallel, immunoprecipitation of pro- caspase-3-myc from HeLa cells co-transfected with CK2a/|3 and pro-caspase-3-myc with 60 uM TBB or DMSO using anti-myc 9E10 antibodies was performed as a control. Membranes were probed with anti-caspase-3 antibodies. 122 blots probed with anti-HA 3F10 conjugated biotin antibodies. Interaction of pro- caspase-3 and CK2 in cells provided further evidence of a functional relationship; however, to investigate whether pro-caspase-3 was phosphorylated in cells an in vivo [y- 32P] label experiment was carried out. A 32 kDa [y-32P] labeled band corresponding to pro-caspase-3 was identified following immunoprecipitation from HeLa cells incubated with [y-32P] for 4 hrs in the presence of DMSO or 60 uM TBB (Figure 4-4B). Notably, a decrease in [y-32P] incorporation of pro-caspase-3 was observed following TBB treatment compared to DMSO, indicating that the phosphorylation of pro-caspase-3 in cells was associated with CK2 activity. Collectively, identification of the interaction of CK2 and pro-caspase-3 in cells and the presence of CK2-dependent phosphorylation of pro- caspase-3 provides mounting evidence for a role of CK2 in the progression of apoptosis via caspase-3 signaling.

4.4.4 Interference with CK2 activity results in an increase in activation of caspase-3 during apoptosis

The activation of caspase-3 requires cleavage at Asp 175 by caspase-8 or -9 resulting in the generation of the active effector caspase that has been shown to be responsible for the proteolytic cleavage of many proteins during apoptosis, including PARP [3, 17, 23]. To test the effect of inhibition of CK2 on the progression of apoptosis and caspase-3 activation, HeLa cells expressing pro-caspase-3-myc were treated with TBB or DMSO for 12 hrs and assayed for PARP cleavage. Treatment of cells expressing pro-caspase-3-myc with TBB resulted in an increase in cleaved PARP (89 kDa) compared to pro-caspase-3-myc expression alone or TBB treatment of cells not expressing caspase- 3-myc, indicating that inhibition of CK2 facilitated caspase-3-dependent cleavage of PARP (Figure 4-5A). To assess the effect of CK2 phosphorylation on caspase-3 activation, the amount of active caspase-3 in cell lysates treated with TBB or DMSO was determined by quantification of the rate of cleavage of the Ac-DEVD-pNA (P-412) colorimetric substrate [24]. A statistical increase in active caspase-3 as determined by ANOVA P= 0.005 was observed in cell lysates expressing pro-caspase-3-myc treated with TBB compared to DMSO, indicating that loss of CK2 results in an increased activation of caspase-3 (Figure 4-5B). Similarly, an increase in the caspase-3 cleavage 123

IB: PARP A caspase-3 B p-tubulin Cleaved PARP • pro- caspase-3r

Cleaved (17 kDa>-

0-tubuliiJh

^fpi(|H(...|Ji,piili|Hi,.mi4J pro-caspase-3myc - + + CK2a-HA + + + TBB + + D IB: caspase-3 2.5 pro- caspase-3- • "S E z <:15 co 'E Cleaved (17 kDa^

Figure 4-5. Inhibition ofCK2 facilitates the maturation of active caspase-3. (A) HeLa cells were co-transfected with CK2a-HA/HA-CK2|3 and pro-caspase-3-myc with 25 fxM TBB or DMSO for 12 hrs. Western blot analysis was performed with anti-PARP and anti-caspase-3 antibodies. (B) Evaluation of the amount of active caspase-3 (pmol/min/jxg) in HeLa cell lysates expressing pro-caspase-3-myc treated with 25 ^M TBB or DMSO for 12 hrs was determined by calculating the rate of cleavage of the Ac- DEVD-pNA (P-412) colorimetric substrate (see materials and methods). Statistical analysis was performed by ANOVA. (C) Western blot analysis probing membranes with anti-caspase-3 antibodies was performed on HeLa cells transfected with either kinase- inactive CK2a-HA (K68M) or active CK2a-HA. (D) The rate of cleavage of the Ac- DEVD-pNA (P-412) colorimetric substrate and the amount of active caspase-3 (pmol/min/^g) was determined in HeLa cell lysates expressing the kinase-inactive mutant of CK2cc-HA (K68M) or active CK2cc-HA. ANOVA (P< 0.01) was performed to test significance. Error bars represent standard deviation within treatments. 124

(17 kDa) fragment was observed in Western blots probed with anti-caspase-3 antibodies in HeLa cells expressing CK2a-HA (K68M) kinase-inactive mutant and pro-caspase-3 - myc, compared to cells expressing wild-type CK2a-HA and pro-caspase-3-myc (Figure 4-5C). A statistical increase in active caspase-3 was also observed in the CK2o>HA (K68M) kinase-inactive-expressing cells, as determined by ANOVA p=0.00000048 in lysates expressing the CK2 kinase-inactive mutant and pro-caspase-3 compared to lysates expressing functional CK2 and pro-caspase-3 (Figure 4-5D). Therefore, the reduction of CK2 activity in cells facilitated the generation of active caspase-3, thus supporting a role for CK2 phosphorylation in the protection of pro-caspase-3 from caspase-mediated cleavage. Consequently, the protection of pro-caspase-3 from caspase-mediated cleavage by CK2 provides a model by which CK2 negatively regulates caspase-3 signaling at two locations promoting cell survival, (1) the prevention of activation of caspase-3, as well as (2) protection of caspase-3 targets from cleavage once caspase-3 becomes active (Figure 4-6).

4.4 Discussion

Identification of the protection of pro-survival proteins from caspase-mediated cleavage by CK2 phosphorylation has provided evidence for an anti-apoptotic role for CK2 [4, 5]. To investigate the anti-apoptotic role of CK2, inhibition studies of CK2 in HeLa cells were performed, which demonstrated that interference with CK2 activity via chemical inhibitors or RNA interference resulted in spontaneous apoptosis. The induction of caspase-dependent apoptosis in the absence of a death stimulus in inhibitor- treated cells was consistent with a requirement for CK2 activity in the negative regulation of caspase activation that had previously been proposed. Therefore, in the present study, the molecular mechanism by which CK2 functions in the regulation of caspase-mediated cleavage within apoptosis signaling pathways was analyzed.

Following a proteome-wide bioinformatics screen for proteins that possess overlapping CK2 and caspase recognition sequences, caspase-3, one of the key aspartic proteases required for the execution phases of apoptosis was identified. A conserved CK2 phosphorylation site was identified in pro-caspase-3, which in its activated form has been shown to play a vital role in the progression of apoptosis, specifically, via the 125

Role for CK2 in caspase-3 signaling

I ft I Active pro-survival caspase-3 targets

" 'Ul ^|| I Cell Survival

Figure 4-6. A model for the regulation of caspase-3 signaling by CK2. (1) The phosphorylation of caspase-3 targets by CK2 protects pro-survival proteins from caspase- 3 mediated cleavage promoting cell survival and tumorigenesis. (2) Phosphorylation of pro-caspase-3 by CK2 within the caspase activation site protects pro-caspase-3 from caspase-8 and -9 mediated cleavage preventing the generation of active caspase-3 and perpetuating the cell survival signal. 126 proteolytic cleavage of key survival proteins [1, 3, 17, 23, 25]. Examining pro-caspase-3 as a putative target in in vitro studies, demonstrated that CK2-phosphorylated pro- caspase-3 within the caspase activation site at Thrl74 and Serl76 resulting in protection from caspase-8 and-9 cleavage. Activation of pro-caspase-3 from its inactive zymogen form into its active form requires caspase-8 or -9 mediated cleavage at Asp 175, followed by cleavage at Asp28, resulting in the generation of a proteolytically active complex consisting of the 17 kDa and 12 kDa cleavage products [1]. Previous studies using peptides to test the effect of phosphorylation on caspase proteolysis demonstrated that phosphorylation of the PI' position adjacent to targeted Asp abolished hydrolysis by caspases, indicating that addition of a bulky phosphorylation proximal to the Asp sterically prevented cleavage [19]. Therefore, the observation that the protective phosphorylation of pro-caspase-3 by CK2 at the P2 and PI' position adjacent to Aspl75, suggested a novel post-translational mechanism for regulation of caspase-3 activation.

Evidence of CK2-dependent phosphorylation of pro-caspase-3 and the existence of CK2 and pro-caspase-3 in complex within cells, as well as the increase in caspase-3 activation observed following CK2 inhibition, further suggests a role for CK2 directly within the caspase activation pathways. Due to the essential role of caspase-3 in apoptosis and perturbations in caspase activation in most cancers, an understanding of the molecular basis involved in the regulation of the caspase-3 has been the focus of many studies. A number of post-translational modifications have been identified contributing to the regulation of caspase-3, including ubiquitination [22], nitrosylation [20], glutathiolation [21] and protein interactions, most notably interaction with IAPs [17, 26]. Interestingly, association of pro-caspase-3 with Calcineurin B (PP2B) has also been observed where Calcineurin B was shown to potentiate the activation of pro-caspase-3 by promoting its proteolytic maturation [27]. The role of Calcineurin B activity in the promotion of cleavage of pro-caspase-3 has yet to be validated, however the presence of a phosphatase in complex with pro-caspase-3 that facilitates caspase activation supports a role for regulation of pro-caspase-3 cleavage by phosphorylation. Recently, proteomic studies identified PP2B as an interacting partner of CK2, which has previously been shown to de-phosphorylate CK2 targets, supporting a functional interaction network between CK2, PP2B and pro-caspase-3 [28, 29]. Further proteomic studies examining 127 the interacting partners of pro-caspase-3 under various conditions, such as CK2 inhibition or Fas-induced apoptosis will be valuable in determining the plasticity of the relationship between CK2 and pro-caspase-3. The regulation of pro-caspase-3 activation during apoptosis appears to be comprised of a highly complex network of modifications, where phosphorylation of pro-caspase-3 within the activation sequence by CK2 could function in addition to, or in combination with, other modifications in the regulation of caspase-3 activity. Further characterization of pro-caspase-3 phosphorylation by CK2 will be required to determine the relationship between phosphorylation protection and the other regulatory pro-caspase-3 modifications in cells, including the examination of the affect of pro-caspase-3 CK2 phosphorylation site mutants on the rate of apoptosis.

Identification of a direct role for CK2 in the regulation of caspase activation, as well as the documented phosphorylation-dependent protection of caspase targets from cleavage provided mounting evidence for an essential role for CK2 in the negative regulation of apoptosis. Our data suggests that regulation of caspase-3 signaling by CK2 phosphorylation occurs during the activation phase of caspase-3 preventing cleavage by caspase-8 and -9, as well as following caspase-3 activation by protecting caspase-3 targets from cleavage (Figure 4-6). The regulation of CK2 activity during apoptosis remains incompletely defined, however, one could envisage the requirement for a decrease in CK2 activity during the execution stages of apoptosis, thereby alleviating the protection associated with CK2 phosphorylation. Alteration of protein kinase activity by caspase-3 has been documented with a number of kinases, including MST3 and PAK2 [30, 31]. Cleavage of the protein kinase results in the loss or gain of kinase activity representing a mechanism of regulation of signaling pathways. However, further studies are required to characterize the mechanism by which CK2 could be regulated in apoptosis.

Identification of other proteins involved in apoptosis with overlapping CK2 consensus and caspase recognition sequences will be key to comprehensively unraveling the molecular mechanisms by which CK2 protects cells from apoptosis. A number of key proteins involved in apoptosis, including other pro-caspases were identified in our bioinformatics screen searching for CK2 and caspase targets, suggesting a global 128 mechanism of regulation of apoptosis by CK2. A systematic evaluation of the identified targets through functional studies could provide a comprehensive CK2 dependent signaling network involved in regulating caspase-targeted cleavage. The emerging anti- apoptotic role of CK2 via the direct negative regulation of caspase signaling pathways and its prominent role in tumorigenesis reiterate CK2 as a strong candidate for anti-cancer therapeutics. Further characterization and identification of CK2 regulated pro-survival targets in apoptosis could provide novel avenues in cancer drug therapy. 129

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CHAPTERS CONCLUSIONS AND PERSPECTIVES: EVOLVING METHODS TO ELUCIDATE THE MOLECULAR MECHANISMS OF CK2 IN TUMORIGENESIS

5.1 The importance of global protein target identification strategies to investigate the role of CK2 in the development of cancer

CK2 is a highly conserved protein serine/threonine kinase that is ubiquitously distributed in eukaryotes, constitutively active and has been implicated in multiple cellular functions, as well as in tumorigenesis and transformation [1]. Elevated CK2 activity has been associated with the malignant transformation of several tissues, and is associated with aggressive tumor behavior [2]. However, the molecular mechanism by which CK2 promotes cell survival remains incompletely understood. Mounting evidence suggests a role for CK2 in tumorigenesis via the regulation of key oncongenes and tumor suppressor proteins within various pro-survival pathways including the Wnt [3], NF-KB [4], and PI3-K pathways [5]. Regulation of oncogene and tumor suppressor proteins by CK2 phosphorylation has been shown to influence susceptibility to proteosome degradation, protect proteins from caspase-mediated cleavage and alter the protein activity [6]. Therefore, elucidation of the molecular mechanisms by which CK2 promotes oncogenesis will require global strategies, including the comprehensive identification of in vivo CK2 substrates. Classically, identification of the majority of CK2 substrates has been achieved using a focused approach, where CK2 and the putative target are specifically analyzed for presence or absence of a CK2-dependent phosphorylation event. The functional relevance of the phosphorylation by CK2 is then analyzed for a change in the biological activity that is being studied, most often through the inhibition of CK2 via chemical inhibitors. However, with the growing number of CK2 targets being identified and the multiple cellular functions attributed to CK2, it is evident that a global systematic evaluation of in vivo CK2 targets in cells is required.

A functional proteomic approach consists of the use of protein separation techniques, in conjunction with mass spectrometry (MS), to identify proteins that change following a specific treatment. This approach has recently been used with success to 135 identify G2/M targets in the MAP kinase pathway, where changes in post-translational modifications (PTM) were observed and identified via MS following treatment with a MKK1/2 inhibitor [7]. The use of kinase inhibitors to manipulate specific kinase activity within a given pathway, in conjunction with MS, provides a tool to investigate the phosphorylation event related to the kinase of interest. This powerful tool allows the systematic dissection of various signaling pathways that contain the target protein kinases and provides information on potential cross-talk between pathways. Identification of changes in proteome profiles following treatment with a specific inhibitor of interest using 2D gel electrophoresis provides information on inhibitor-dependent protein expression and phosphorylation changes. However, the use of kinase inhibitors to identify in vivo substrates using functional proteomics presents the limitation of relying on the inhibitor being specific for only the kinase of interest. Off-target effects of the inhibitors may lead to identification of inhibitor-dependent changes that are independent of the kinase being studied. Therefore, a systematic evaluation of the inhibitors being utilized in functional proteomics experiments is required, as well as employing strategies to validate that changes in phosphorylation observed are due to inhibition of target kinase. The specificity of current CK2 inhibitors and the efficiency to inhibit CK2 in vivo has not been systematically characterized. Therefore, the research presented in Chapter 2 focused on elucidating the mechanism of action of CK2 inhibitors in cells, including compounds currently being used in pre-clinical studies. Development of strategies to address inhibitor specificity, including the use of inhibitor-resistant CK2 mutants to rescue inhibitor-dependent cellular effects were explored. A greater understanding of the specificity of CK2 inhibitors in cells provides vital information regarding the use of the inhibitors to identify CK2 substrates using functional proteomics, as well as in the evaluation of the inhibitors as potential anti-cancer therapeutics. 136

5.2 Identification of CK2 inhibitor off-targets using chemo-proteomics: A re- evaluation of the use CK2 inhibitors to study CK2 function

Elevated CK2 activity has been established in a number of cancers, where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins [6]. Consequently, the development of CK2 inhibitors has been ongoing in pre-clinical studies, resulting in the generation of a number of CK2- directed compounds. The crystal structure of the CK2 haloenzyme has been solved, providing insights into drug design and methods to increase specificity [8, 9]. Unique structural properties of the CK2a and CK2a' catalytic ATP binding pocket, mainly its shape and reduced size, have allowed for the development of highly selective CK2 inhibitors [10]. In Chapter 2, an unbiased evaluation of commercially-available protein kinase CK2 inhibitors using a chemo-proteomic approach was carried out. Treatment of human cancer cells with CK2 inhibitors TBB, TBBz and DMAT resulted in the induction of apoptosis. However, inhibitor-specific cellular effects were observed with each of the inhibitor derivatives suggesting that the inhibitors exhibited unique modes of drug action. Furthermore, employment of inhibitor-resistant CK2 mutants to investigate the apoptotic effect associated with the inhibitors demonstrated that restoration of CK2 activity in inhibitor-treated cells was not sufficient to rescue the observed apoptosis. These findings suggested that the inhibitors were potentially interfering with other essential cellular processes resulting in subsequent cell death. Therefore, a global analysis of inhibitor protein interactors was undertaken to determine off-targets utilizing a chemo-proteomic approach, as described in Chapter 2. As expected, initial studies to determine whether the inhibitors interacted with CK2 demonstrated that TBB, TBBz and DMAT were all capable of binding to both CK2a and CK2a'. Once CK2 was validated as a bona fide target of the CK2 inhibitors, a global analysis of all potential inhibitor-interacting ATP binding proteins was undertaken. Utilizing ATP-affmity chromatography in conjunction with 2D gels and mass spectrometry, a number of putative inhibitor interactors were identified, including Quinone Reductase 2 (QR2). The discovery of non-kinase ATP-binding proteins interacting with CK2 inhibitors reiterated the importance of using a global method to test kinase specificity that 137 encompasses all ATP-binding proteins. QR2 has been shown to function in the detoxification processes of quinones, specifically, the two-electron reduction of menadione by the oxidation of N-alkylated or N-ribosylated nicotinamides [11]. Recently, studies investigating the function of QR2 in cells showed that inhibition of QR2 in keratinocytes resulted in suppressed survival and increased TNF-induced apoptosis [12]. Other studies investigating the mechanism of action of ABL and PKC kinase inhibitors demonstrated QR2 as a novel drug target, suggesting a role for QR2 in the metabolism of a variety of compounds in the cell [13, 14]. Further studies to determine the mechanism by which these CK2 inhibitors interact with QR2, as well as the effect of the inhibitors on QR2 function in cells will be required. Interestingly, QR2 has been identified as a key drug target of the anti-malarial compound, Quinacrine, where inhibition of QR2 is thought to alter the redox status of the infected red blood cells resulting in death [15]. Therefore, it would be of particular interest to evaluate the efficacy of TBBz and DMAT to inhibit QR2, as they might present as novel anti-malarial compounds. The identification of novel CK2 inhibitor-binding proteins provides insight into the mode of drug action of the inhibitors, which will contribute in the development of more specific next-generation CK2 inhibitors. The chemo-proteomic approach can be applied in the evaluation of a variety of ATP analog inhibitors, and therefore offers a novel strategy to evaluate protein kinase drug specificity. The unbiased evaluation of CK2 inhibitors provided evidence of inhibitor off-targets, however, due to the prominent role of CK2 in tumorigenesis and its emerging anti-apoptotic role, inhibition of this kinase remains a promising candidate for anti-cancer therapeutics. Following the identification of CK2 inhibitor off-targets using an unbiased chemo-proteomic approach, it is evident that validation strategies will be required when using the inhibitors to globally identify CK2 substrates in cells. With the development of CK2 inhibitor-resistant mutants that are capable of restoring CK2 function in cells, comes the intriguing possibility of identifying and validating global CK2 targets using a functional proteomic approach (Figure 5-1). The identification of bona fide CK2 targets would consist of an evaluation of changes in the CK2-dependent phospho-proteome at different stages of the cell cycle in the presence of CK2 inhibitors. Changes in the incorporation of 32P in the presence of CK2 inhibitors would be identified 138

Observe phosphorylation changes Validation of targets Control Rescue experiments using inhibitor resistant mutants

* Bioinformatics • Database Search

Protein Identification + CK2 Mass spectrometry inhibitor

32P labeling, Fluorescence stains, phospho-specific antibodies Identify CK2 pathways involved in tumorigenesis

Figure 5-1. Utilizing a functional proteomic approach to identify CK2 substrates in cells. Cells are treated with CK2 inhibitors, followed by separation of proteins using 2D gel electrophoresis. Changes in phosphorylation are detected using various methods, including [y-32P] labeling of cells. Proteins corresponding to phosphorylation changes are then identified using mass spectrometry. Characterization of proteins identified using bioinformatics and database searches are performed, followed by validation using CK2 inhibitor-resistant mutants. 139 using 2D gel electrophoresis and autoradiography in conjunction with mass spectrometry. Rescue experiments validating the CK2 inhibitor-dependent phosphorylation changes would be carried out in 32P-labeled cells expressing wild-type CK2 or inhibitor-resistant CK2. Inhibitor-dependent changes in phosphorylation that are rescued by the inhibitor- resistant CK2 mutants provide strong evidence that the protein is a bona fide CK2 target. However, using a functional proteomic approach to identify phosphorylation targets could result in the potential identification of changes in phosphorylation that occur indirectly from the inhibition of CK2. The change in phosphorylation of an identified CK2 target could be due to an increase or decrease in stimulus of another signaling pathway that is regulated by CK2 activity. Therefore, a detailed characterization of the protein target identified in the functional proteomic screen is required, to address whether it is a direct target of CK2 or whether the phosphorylation is an indirect downstream effect of CK2 inhibition. Preliminary studies using a functional proteomic approach to explore CK2 inhibitor biomarkers was undertaken to systematically identify putative CK2 substrates in cells. A number of changes in [y-32P] incorporation were observed in inhibitor-treated HeLa cells, indicated by a decrease or loss of [y-32P] at distinct spot locations on 2D gels exposed to autoradiography compared with DMSO controls (Appendix B, Figure B-l). A number of previously identified CK2 substrates were detected in this study including, CPa, Nm23, nucleophosmin and EF-1 delta (EEF1D) (Appendix B, Table B-l). The detection of changes in [y-32P] incorporation of known CK2 targets in cells suggests that the inhibitors will be useful in the identification of CK2 inhibitor-dependent substrates, as well as in the investigation of the molecular mechanisms of CK2 within signaling pathways. Interestingly, EEF1D, a protein involved in the elongation steps of mRNA translation was identified to show a decrease in [y-32P] incorporation following CK2 inhibitor treatment in HeLa cells. Previous studies have shown that CK2 phosphorylates rabbit EEF1D in vitro, however, the functional consequence of the phosphorylation remains incompletely understood [16-18]. Studies investigating the role of EEF1D in cancer demonstrated that higher expression of EEF1D correlated with metastasis, advanced disease stages and poor prognosis for patients, suggesting EEF1D as a potential 140 anti-cancer therapeutic target [19]. Further studies to determine the effects of CK2 phosphorylation on human EEF1D function are required, first and foremost of which is the determination of EEF1D as a bona fide CK2 target in cells. Notably, the predicted CK2 phosphorylation site in EEF1D has been identified in large-scale proteomics studies as being phosphorylated in cells, reiterating the importance of studying the relationship between EEF1D and CK2 [20]. Preliminary studies using tandem affinity purification techniques demonstrated that CK2a and EEF1D interacted in cells suggesting that CK2 may be involved in the phosphorylation of EEF1D (data not shown). However, rescue experiments employing inhibitor-resistant mutants will be required to validate EEF1D as a bona fide CK2 target in cells, as well as in evaluating the functional consequence of CK2-EEF1D interactions. Overall, identifying CK2 phosphorylation targets using a functional proteomic approach that contribute to tumorigenesis will help reconcile the mechanism by which CK2 functions in the protection of cells from apoptosis, as well as resolve a role for CK2 in molecular cross-talk with various signaling pathways. The identification of CK2 inhibitor off-targets using chemo-proteomics, reiterated the necessity for strategies to validate inhibitor-dependent changes in phosphorylation as CK2-dependent. Employment of inhibitor-resistant mutants in conjunction with the functional proteomics approach will provide a global strategy to systematically identify CK2 substrates in cells. It will be of importance to identify CK2 targets that bring together multiple signaling events that are essential for normal growth and development of the cell. Understanding perturbations of regulatory proteins, specifically CK2, within these signaling networks will allow for the development of rational anti-cancer therapeutic strategies.

5.3 Generation of analog-sensitive CK2 gatekeeper mutants to identify global CK2 targets The potential for identifying indirect and off-target phosphorylation changes using CK2 inhibitors in conjunction with a functional bioinformatic approach, reiterates the requirement for a strategy that can directly identify kinase targets. A chemical genomics approach that utilizes a mutant protein kinase that is capable of binding an ATP-analog, as described by Shokat and colleagues, could also be used in combination with the 141 functional proteomic approach to identify bona fide CK2 targets [21, 22]. A mutation in a bulky hydrophobic group in the ATP binding pocket termed the "gatekeeper" results in a catalytically-active analog-sensitive (AS) protein kinase that is capable of binding a larger ATP analog. This strategy provides opportunities to identify direct targets of a specific kinase in the presence of all other cellular protein kinases. The gatekeeper residue within CK2 was identified via comparison with protein kinases that have successfully been utilized in the Shokat method to identify substrates. Specifically, alignment of CK2 with CDK7, a kinase in the same CMGC family as CK2, showed a conserved bulky residue at Phel 13 for CK2a and Phel 14 for CK2a' (Figure 5- 2). Mutation of Phe91->Gly in CDK7 generated an analog-sensitive mutant that was used to identify a number of novel substrates, supporting the use of CK2 gatekeeper mutants in the systematic identification of CK2 targets [23]. Generation of CK2 analog- sensitive mutants was undertaken via mutation of the Phel 13 (CK2a) and Phel 14 (CK2a') gatekeeper to Ala or Gly using site-directed PCR mutagenesis. Characterization of the functional consequence of mutating the gatekeeper residue in CK2 was evaluated by assessing protein expression and kinase activity in cells. The CK2a-HA (Fl 13G) and HA-CK2a'(F114A) gatekeeper mutants were shown to express, as well as retain kinase activity in cells as evident by the presence of autophosphorylation of CK2|3 following immunoprecipitations of the mutants (Appendix B, Figure B-2). However, further studies characterizing the CK2a-HA(F113G) and CK2a'(F114A) mutants in cells will be required. The demonstration that CK2o>HA (F113A) and HA-CK2a'(F114A) mutants both express and are functional in cells supports the use of the gatekeeper mutants to identify CK2 global substrates using the Shokat method. A variety of bulky N6-modified ATP-analogs have been synthesized to be used with analog-sensitive protein kinases, including N6-benzyl ATP, N6-phenylethyl ATP and N6-cyclopentyl ATP [24]. Classically, [y-32P] labeling of the y-phosphate of the N6- modified ATP-analogs is carried out and incorporation of [y- P] into substrates is used to identify protein targets [25]. However, due to limitations in the purification of [y-32P] labeled proteins, alternative strategies utilizing non-radioactive y-phosphate labels, including biotin were explored. Previous studies have demonstrated that CK2 was capable of using biotin-labeled y-phosphate ATP to phosphorylate a known in vitro target Alignment of gatekeeper residue in CK2 and CDK

CDK4 REIKVTLVFEHVD-QDLRTYLDKAP CDK6 RETKLTLVFEHVD-QDLTTYLDKVP CDK7 HKSNISLVFDFME-TDLEVIIKDNS CK2ct' VSKTPALVFEYINNTDFKQLY CK2a VSRTPALVFEHVNNTDPKQLY

Figure 5-2. Alignment of gatekeeper residue in CK2a/a' with CDK family members. 143

[26]. The use of a non-radioactive ATP analog in a chemical genetic approach represents an intriguing strategy to identify CK2 substrates. Identification of global CK2 substrates utilizing the CK2-AS mutants with biotin- labeled y-phosphate N6-benzyl ATP will provide a systematic strategy to validate existing CK2 targets as bona fide CK2 substrates, as well as identify novel CK2 substrates. Using biotin-labeled y-phosphate as the method for identifying phosphorylated targets offers an efficient method for purifying putative targets from cell lysates via affinity purification with streptavidin. Lysates of cells expressing CK2-AS mutants or the use of purified GST-CK2-AS incubated with biotin-labeled y-phosphate N6-benzyl ATP can be utilized in conjunction with functional proteomics and/or immunoprecipitations to identify or validate CK2 targets (Figure 5-3). A large number of CK2 substrates have been identified, however, few have been validated as bona fide substrates [27]. The use of streptavidin affinity capture techniques to isolate proteins that incorporate biotin-labeled Y-phosphate, followed by protein identification by mass spectrometry or the use of antibodies will provide a strategy to comprehensively identify CK2 substrates. However, preliminary studies are required to determine the efficiency and stability of biotin-labeled Y-phosphate reactions in cell lysates. Initial studies using CK2 peptide sequences to test the ability of the CK2-AS to utilize the biotin-labeled y-phosphate N6-benzyl ATP will be performed, where biotin incorporation will be assessed using mass spectrometry. Following validation that CK2-AS mutants are efficient at using biotin-labeled y- phosphate N6-benzyl ATP as a substrate, large-scale functional proteomics experiments will be carried out. However, using biotin-labeled y-phosphate N6-benzyl ATP analogs presents a limitation, in that the large bulky ATP analogs cannot penetrate intact cells. Therefore, subsequent experiments will be required to evaluate the putatively identified CK2 targets in cells using classical methods, such as inhibitor studies. The combination of functional proteomics and inhibitor-resistant rescue experiments, along with analog-sensitive CK2 mutants could provide a comprehensive identification of cellular CK2 targets (Figure 5-1 and 5-3). The ability to use isoform- specific inhibitor-resistant and analog-sensitive CK2 mutants will also provide essential information on the functional specialization of CK2. The comparison of the two approaches will offer insight into the molecular mechanism of action of CK2 inhibitors 144

GST-CK2-GM or HA-CK2-GM Readout: Biotin-Streptavidin p> • Streptavidin-conjugated beads • Streptavidin-coated plates °-< • Anti-Biotin antibodies 1 t« PG

Biotin-Y-Phosphate- N6-Benzyl-ATP

Functional proteomics mmunoprecipitations (global targets) (specific targets)

Discovery Validation

MALDI/TOF Antibodies "protein identification" "protein identification"

Figure 5-3. The use of a chemical genetic approach in conjunction with functional proteomics to identify CK2 substrates. Biotin-labeled y-Phosphate N6-Benzyl-ATP is added to cell lysates containing HA-CK2-GM or GST-tagged CK2-GM gatekeeper mutants. Following incubation, biotin-labeled proteins will be isolated using affinity chromatography using streptavidin-conjugated sepharose. Protein identification will be performed using mass spectrometry or via the use of antibodies. 145 through the investigation of differences in the phospho-proteome profiles. If phosphorylation events change in the presence of the inhibitor, but not with the analog- sensitive CK2 mutants, then it raises the possibility that the phosphorylation change is due to the indirect regulation by CK2 or is a potential off-target effect of the inhibitor. The use of functional proteomics in combination with chemical genetics will provide a comprehensive strategy to evaluate CK2 targets, where a bona fide CK2 substrate will require identification by both strategies.

5.4 Convergence of protein kinases and caspase signaling pathways: Evidence for a global role of phosphorylation in the regulation of caspase signaling

The convergence of protein kinases and caspase signaling pathways has recently become evident, as phosphorylation of a number of caspase substrates at or near the caspase recognition motif has been shown to prevent caspase-mediated cleavage [1]. Specifically, phosphorylation of a residue at the PI' position adjacent to the cleaved aspartic residue has been shown to abolish caspase hydrolysis [28]. Evidence of a structural mechanism for phosphorylation-dependent protection of caspase substrates, as well as the protection of a wide variety of caspase targets by multiple protein kinases, suggests a global role for phosphorylation in the negative regulation of apoptosis. The constitutively active protein kinase CK2, has been implicated in the protection of multiple caspase substrates from caspase-mediated cleavage, as well as shares an overlapping requirement with caspases for an acidic consensus sequence. Therefore, to investigate the global role of phosphorylation in caspase signaling, a comprehensive evaluation of overlapping proteins with a CK2 phosphorylation and a caspase recognition motif was carried out in Chapter 3. To evaluate phosphorylation as a global mechanism of regulation of caspase signaling pathways, a novel sequence-based protein identification targeting strategy (sPITS) was developed. The strategy consisted of combining the global strengths of bioinformatics with the throughput capacities of peptide array phospho-target screens and fluorescent-based caspase substrate identification assays. Following a proteome-wide bioinformatics search, over 300 proteins were identified that possessed overlapping 146

CK2/caspase recognition motifs, supporting a role for CK2 in the global protection of targets from caspase-mediated cleavage. Intriguingly, 11% of the proteins indentified had been shown previously to be phosphorylated at the predicted CK2 site in cells. Although rigorous identification of the kinase responsible has yet to be determined, our data is consistent with the involvement of CK2. A number of peptides corresponding to proteins containing an overlapping CK2/caspase consensus were shown to be phosphorylated in the CK2 peptide array target screens, as well as cleaved by caspases using Caspase Substrate Identification (CSI), representing novel candidate CK2/caspase targets. In addition to its ability to identify caspase substrates, this technique provides a platform to test protease activity with the potential for high throughput capacity. The exploitation of the CSI will be highly advantageous for the study of protease substrate recognition, effect of post-translational modifications on proteolysis, as well as in the detection of protease activity. The use of bioinformatics in combination with throughput strategies such as peptide array phospho- target screens and CSI offers a powerful tool for the identification of widespread protein kinase and/or protease substrates. However, further studies to evaluate the candidate CK2/caspase substrates as bona fide CK2 and caspase targets in cells will be required. The use of previously established strategies, such as inhibitor-resistant and analogue sensitive CK2 mutants will be beneficial in the identification of CK2/caspase candidates as bona fide targets in cells. The sensitivity of the CK2/caspase substrates to caspase cleavage during apoptosis can be investigated via the assessment of protein stability following induction of apoptosis through western blot analysis.

5.5 Regulation of caspase-3 proteolytic activation by CK2: a mechanism for protection of cells from apoptosis

Pro-caspase-3 was identified as a putative CK2 target following a proteome-wide search for proteins that contain a CK2 consensus sequence for phosphorylation within a caspase cleavage site. Pro-caspase-3, a well-documented effector caspase involved in the execution phase of apoptosis, requires proteolytic activation via cleavage at Asp 175 by caspase-8 and/or -9 [29]. Once activated, caspase-3 proteolytically cleaves multiple 147 targets involved in cell survival leading to the demolition of the cell. Identification of a highly conserved CK2 site at the P2 and PI' residue adjacent to Asp 175 suggests a role for CK2 in the regulation of caspase-3 activation. In Chapter 4, phosphorylation of pro- caspase-3 at Thrl74 (P2) and Serl76 (PI') by CK2 was observed in in vitro kinase assays, where the phosphorylation protected pro-caspase-3 from caspase-8 and -9 mediated cleavage. An evaluation of pro-caspase-3 and CK2 interaction in cells showed that CK2 and pro-caspase-3 exists in a complex where pro-caspase-3 was phosphorylated in a CK2-dependent manner. Treatment of cells with CK2 inhibitors or expression of kinase inactive CK2 mutants resulted in an increase in cleaved pro-caspase-3 suggesting that CK2 activity was required for stabilization of pro-caspase-3 in cells. Regulation of pro-caspase-3 activation by CK2 phosphorylation represents a novel mechanism by which CK2 protects cells from apoptosis, via direct regulation of caspase signaling. To further characterize the role of CK2 in the regulation of caspase-3 activation, cellular studies using the pro-caspase-3 (T174A and S176A) phosphorylation site mutants were performed. Notably, the mutants were shown to be resistant to caspase-mediated cleavage following etoposide treatment in cells (Appendix B, Figure B-3). These findings suggest that mutation of the Thrl74 and Serl76 to Ala interfered with the mechanism of recognition and cleavage by cellular caspase-8 and/or -9. The ability of caspase-9 to cleave pro-caspase-3 mutants (T174A and SI76A) in vitro but not in vivo suggest that altering the caspase recognition site interferes with other molecular events required for the cleavage of pro-caspase-3. Recently, studies investigating the sequence specificity of other caspases for targets showed that both caspase-8 and -9 favored a Val at the P2 position, while caspase-8 preferred a Gly at the PI' residue, while caspase-9 preferred a Thr at PI' [30]. Therefore, utilizing engineered pro-caspase-3 phosphorylation mutants with the Thr 174 -> Val and the Serl76 -> Gly should provide a more attractive substrate for cellular caspase-8 and/or -9 in future studies. An evaluation of the molecular consequences of removing the CK2 phosphorylation sites on the proteolytic activation of pro-caspase-3 will provide valuable information on the molecular mechanism by which CK2 functions in caspase signaling. However, the regulation of pro-caspase-3 activation during apoptosis appears to be comprised of a highly complex network of modifications, where phosphorylation of pro-caspase-3 within the activation sequence by CK2 could 148 function in addition to, or in combination with, other modifications in the regulation of caspase-3 activity. In the present studies, a model has been proposed for a role for CK2 in the regulation of caspase signaling where CK2 functions directly within the caspase signaling cascade by preventing activation of caspase-3, as well as indirectly via the protection of caspase targets from caspase-mediated cleavage. The regulation of CK2 activity during apoptosis remains incompletely defined, however, one could envisage the requirement for a decrease in CK2 activity during the execution stages of apoptosis, thereby alleviating the protection associated with CK2 phosphorylation. Intriguingly, upon analysis of the effect of phosphorylation of pro-caspase-3 by CK2 it was noted that pre-incubation of CK2 with active caspase-3 resulted in the cleavage of CK2 (Appendix B, Figure B-4). Alteration of protein kinase activity by caspase-3 has been documented with a number of kinases, including MST3 and PAK2 [31, 32]. Cleavage of the protein kinase results in the loss or gain of kinase activity, representing a mechanism of regulation of signaling pathways during apoptosis. Cleavage of CK2 by caspase-3 could provide a novel mechanism for the negative regulation of CK2 during apoptosis; as well as provide a method by which caspase-3 could regulate its own activation. However, further studies are required to characterize the physiological effect of cleavage of CK2 by caspase-3; however, evidence for a negative feedback mechanism between CK2 and caspase-3 could provide an attractive model for studying the regulation of apoptosis by CK2. Regulation of pro-caspase-3 proteolytic activation, as well as the protection of a number of targets from caspase cleavage by CK2 phosphorylation is thought to contribute to the prevention of apoptosis during tumorigenesis. Presumably, under oncogenic conditions, elevated CK2 levels continually phosphorylate the proposed caspase targets preventing cleavage and promoting cell survival. However, under non-oncogenic conditions, removal of the protective phosphorylation must occur to alleviate the repression allowing the progression of apoptosis. This suggests that a phosphatase may function in collaboration with CK2 in the regulation of caspase-signaling, where CK2 inhibits caspase cleavage, while the phosphatase promotes it. Interestingly, association of pro-caspase-3 with the protein phosphatase Calcineurin B (PP2B) has also been observed where Calcineurin B was shown to perpetuate the activation of pro-caspase-3 by 149 promoting its proteolytic maturation [33]. Proteomic studies have identified PP2B as an interacting partner of CK2, which has previously been shown to de-phosphorylate CK2 targets, supporting a functional interaction network between CK2, PP2B and pro-caspase- 3 [34, 35]. Taken together, the studies suggest a role for protein phosphatase PP2B and CK2 in the regulation of pro-caspase-3 activation, where pro-caspase-3, CK2 and PP2B exists in a protein complex, favoring phosphorylation or de-phosphorylation depending on cellular signals. Proteomic studies examining the interacting partners of pro-caspase-3 under various conditions, such as CK2 inhibition or Fas-induced apoptosis will be valuable in determining the plasticity of the relationship between CK2 and pro-caspase-3. A comprehensive characterization of pro-caspase-3 interacting partners in cells, specifically investigating potential interactions with protein kinases and phosphatases, will be essential in determining the molecular mechanisms involved in the regulation of caspase signaling. The use of proteomic and mass spectrometry strategies such as SILAC, could provide a powerful tool to quantitate changes in pro-caspase-3 interacting partners within caspase signaling pathways during the progression of apoptosis (Figure 5- 4). The acquired knowledge of the diversity and dynamics of proteins that interact with pro-caspase-3 in cells during survival and cell death will be essential in elucidating the molecular mechanisms by which CK2 functions in the regulation of caspase signaling.

5.6 Perspectives: A working model for CK2 in the negative regulation of caspase signaling Mounting evidence suggests an anti-apoptotic role for CK2 via the negative regulation of caspase signaling, via the prevention of activation of caspase-3, as well as the protection of survival targets from caspase-mediated cleavage. These findings offer an attractive model explaining how elevated CK2 levels contribute to transformation and tumorigenesis. However, further studies elucidating the molecular mechanisms by which CK2 promotes cell survival via the negative regulation of caspase signaling are required. Convergence of strategies designed to identify CK2 targets such as the use of CK2 inhibitors in conjunction with inhibitor-resistant and analog-sensitive CK2 mutants will be crucial for the validation of the large number of candidate CK2/caspase targets identified in the present sPITS studies. Of particular interest, will be the 150

FasL TNFa No Treatment ..IBB-

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Cells grown in light isotope- Cells grown in heavy isotope- containing media containing media

IP: Myc Harvest and lyse cells Stain: Gelcode blue — ?4» Immunoprecipitation Pro- — **•» «n> caspase-3- myc • z: — : Y Trypsin digestion on beads

LC-MS/MS

0) Ratio Determination

m/z Light Heavy

Figure 5-4. The use ofSILAC to identify the dynamic relationship between CK2 and caspases during apoptosis. Cells grown in light isotope-containing media are treated with apoptotic stimulus and/or CK2 inhibitors, while cells grown in heavy isotope media are used as no treatment controls. Cells are mixed and harvested, followed by immunoprecipitation of the protein of interest, trypsin digested and subjected to LC- MS/MS. Changes in relative intensity of MS peaks between the light and heavy isotope treatments will be analyzed and a ratio determined. 151 characterization of the role of CK2 phosphorylation and caspase cleavage in the regulation of the apoptosis inhibitor protein Aven, as well as a number of transcriptional regulators, including CHD8. Identification of pro-caspase-8 in the functional bioinformatics screen as a putative CK2/caspase target, and the validation that phosphorylation by CK2 protects pro- caspase-3 from cleavage, suggests a role for CK2 in protection of pro-caspase-8. The proteolytic activation site sequence within pro-caspase-8 contains a CK2 consensus site for phosphorylation at the P2 and PI' residues identical to pro-caspase-3 (VETDSEE). Therefore, one can envisage the phosphorylation of pro-caspase-8 at the P2 and PI' residues preventing the proteolytic activation of caspase-8, and subsequent activation of caspase-3. The possibility for protection of pro-caspase-8 from proteolytic activation by CK2 presents an intriguing mechanism by which CK2 could regulate the caspase signaling pathway at both the initiation and execution phases of apoptosis. Notably, phosphorylation of active caspase-9 was observed by CK2 in in vitro experiments testing the effect of CK2 phosphorylation on caspase cleavage (data not shown). This provides further evidence for a role for CK2 in the regulation of the caspase signaling, however, experiments are required to determine the effect of CK2 phosphorylation of caspase-9 on substrate specificity and cleavage. Further examination of the molecular components involved in the proteolytic activation of caspase-3 revealed a number of proteins associated with CK2 that have been implicated as caspase targets including, Aven, Bad, Apaf-1, caspase-8 and caspase-9 [36-39]. It will be of interest to determine whether CK2 phosphorylates or interacts with these apoptotic proteins, as well as to investigate further the role of CK2 in the regulation of the initiation phase of apoptosis.

5.7 Conclusion In summary, elevated CK2 activity has been established in a number of cancers, where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently, the development of CK2 inhibitors has been ongoing in pre-clinical studies, resulting in the generation of a number of CK2-directed compounds. Therefore, an unbiased evaluation of commercially- available CK2 inhibitors was carried out using a chemo-proteomic approach. Utilizing 152

ATP-affinity chromatography in conjunction with 2D gels and mass spectrometry, a number of putative CK2 inhibitor off-targets were identified. The chemo-proteomic approach can be applied in the evaluation of a wide variety of ATP-analog inhibitors, offering a systematic strategy to evaluate protein kinase inhibitor specificity. Identification of CK2 inhibitor off-targets demonstrates that strategies will be required to validate cellular effects observed using CK2 inhibitors, such as employing inhibitor- resistant and analog-sensitive CK2 mutants. The convergence of protein kinases and caspase signaling pathways has become increasingly evident, as phosphorylation of caspase substrates within the caspase recognition motif has been shown to prevent cleavage. Interestingly, a strong similarity exists between the caspase degradation recognition sequence and the CK2 consensus motif for phosphorylation, signifying that CK2 may be involved in protection of a wide variety of caspase targets. Therefore, to investigate the global role of phosphorylation in caspase signaling, a comprehensive evaluation of overlapping proteins with a CK2 phosphorylation and a caspase recognition motif was carried out. The identification of numerous novel CK2/caspase targets in the functional bioinformatics studies, taken together with the previously identified caspase targets protected by CK2 phosphorylation, provides mounting evidence for a global role for phosphorylation in the regulation of caspase signaling. The combination of the protection of pro-survival proteins from caspase-mediated cleavage and promotion and repression of proteins within apoptosis signaling pathways suggests an essential role for CK2 in the promotion of cell survival (Figure 5-5). In the present studies, phosphorylation of pro-caspase-3 by CK2 was shown to regulate caspase-3 activity via the inhibition of proteolytic activation. Previous studies have demonstrated that CK2 negatively regulates the proteolytic activation of pro- caspase-9, as well as prevents the activating dimerization of caspase-2 during apoptosis. Evidence from the present studies support a role for CK2 in the regulation of the proteolytic activation of pro-caspase-8, as well as in the stabilization of the anti-apoptotic protein, Aven. Taken together, the prevention of activation of pro-caspase-3, -9 and the putative protection of pro-caspase-8 by CK2 phosphorylation establishes an essential role for CK2 in the regulation of the progression of caspase cascades. CK2 has also been shown to contribute in the stabilization of transcription factors, thereby promoting the 153

DMth Stimulus 0*.TNF,FuL) .f•€*«**

CK2/c—paaa trprt | Putattwa protection of CK2/ Direct stimulatory affect wpiii target from Putative phosphorylation target

Phosphatase Protection of CK2/caspase -4 Direct MtMtory effect target from CHPHI 1 • «• Phosphorylation, unknown •Had Transcription factor mediated dMwy

Figure 5-5. The global role of phosphorylation in the regulation ofcaspase signaling. The regulation of activation of caspases, as well as the prevention of cleavage of a variety of caspase targets by CK2, illustrates the multi-disciplinary role for CK2 in the regulation ofcaspase signaling. 154 expression of pro-survival signals, such as Survivin and c-Myc. Identification of a wide variety of transcription factors as putative CK2/caspase targets, including CHD's and HDAC4, supports a role for CK2 in transcriptional regulation. Overall, evidence presented here strongly suggest that CK2 plays an essential role in the negative regulation of caspase signaling pathways, via the global protection of caspase substrates from caspase-mediated cleavage. The emerging anti-apoptotic role of CK2 via the direct negative regulation of caspase signaling pathways and its prominent role in tumorigenesis reiterates CK2 as a strong candidate for anti-cancer therapeutics. 155

5.8 Reference:

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APPENDIX A

Table A-l. Proteins identified using a peptide match program that contained an overlapping CK2 consensus for phosphorylation and a caspase cleavage recognition motif. SwissProt and NCBI databases were used for the peptide match program.

Peptide Match SwissProt Protein Name DE.D[ST]..[DE]

MPP10_HUMAN U3 small nucleolar O00566 sdeditnvhdDELDSNKEddeiaeeeae ribonucleoprotein protein MPP10 - Homo sapiens

PLD2_HUMAN Phospholipase D2 - 014939 ldssqlqraesDEVDTLKEgedpadrmhp Homo sapiens

K0406_HUMAN Uncharacterized 043156 eeeqsvppkvDENDTRPDvepplplqiq protein KIAA0406 - Homo sapiens

SPY1_HUMAN Portein sprouty 043609 kgifyhcsndDEGDSYSDnpcscsqshc homolog 1 - Homo sapiens

075558 qydqqfpdgdDEFDSPHEdivfetdhil STX11_HUMAN Syntaxin-11 - Homo sapiens

CBPDHUMAN Carboxypeptidase D 075976 kksllshefqDETDTEEEtlyssk precursor - Homo sapiens

094915 plptarrhdeDEDDSLKDrelmvtsrrw FRYL_HUMAN Protein furry homolog-like - Homo sapiens

TBG1_HUMAN Tubulin gamma-1 P23258 rkedmfkdnfDEMDTSREivqqlideyh chain - Homo sapiens

RFC2_HUMAN Replication factor C P35250 pkgrhkiiilDEADSMTDgaqqalrrtm subunit 2 - Homo sapiens

INAR2_HUMAN Interferon- P48551 kkkvwdynydDESDSDTEaaprtsgggy alpha/beta receptor beta chain precursor - Homo sapiens

P49427 eeeadscfgdDEDDSGTEe

NEK3_HUMAN Serine/threonine- P51956 ldperlepglDEEDTDFEeeddnpdwvs protein kinase Nek3 - Homo sapiens

IF39_HUMAN Eukaryotic translation P55884 rlelrggvdtDELDSNVDdweeetieff initiation factor 3 subunit 9 - Homo sapiens

RAG2_HUMAN V(D)J P55895 saeansfdgdDEFDTYNEddeedesetg recombination-activating protein 2 - Homo sapiens

ICBR_HUMAN Caspase-1 inhibitor P57730 isqedmnkvrDENDTVMDkarvlidlvt Iceberg - Homo sapiens

3BP2_HUMAN SH3 domain-binding P78314 ptdnedyehdDEDDSYLEpdspepgrle protein 2 - Homo sapiens

P98161 fslvakrlhpDEDDTLVEspavtpvsar PKD1_HUMAN Polycystic 1 precursor - Homo sapiens

BAXAHUMAN Apoptosis regulator Q07812 klseclkrigDELDSNMElqrmiaavdt BAX, membrane isoform alpha - Homo sapiens

BAXBHUMAN Apoptosis regulator Q07814 klseclkrigDELDSNMElqrmiaavdt BAX, cytoplasmic isoform beta - Homo sapiens

NFIAJHUMAN Nuclear factor 1 A- Q12857 sstkrlksveDEMDSPGEepfytgqgrs type - Homo sapiens

DMP1_HUMAN Dentin matrix acidic Q13316 tgkggddkddDEDDSGDDtfgdddsgpg phosphoprotein 1 precursor - Homo sapiens

CV019_HUMAN Uncharacterized lfkppedsqdDESDSDAEeeqttkrrrp Q13769 protein C22orfl 9 - Homo sapiens

GNRP_HUMAN Guanine nucleotide- Q13972 vsssftnkipDEGDTTPEkpedpsalsk releasing protein - Homo sapiens

PTCA_HUMAN Protein tyrosine Q14761 stdndlerqeDEQDTDYDhvadgglqad phosphatase receptor type C- associated protein - Homo sapiens

NOLCl_HUMAN Nucleolar Q14978 qpvessedssDESDSSSEeekkpptkav phosphoprotein pi30 - Homo sapiens

IF4H_HUMAN Eukaryotic translation Q15056 kfkgfcyvefDEVDSLKEaltydgallg initiation factor 4H - Homo sapiens

VPS72_HUMAN Vacuolar protein Q15906 eyqgdqsdteDEVDSDFDidegdepssd sorting-associated protein 72 homolog - Homo sapiens

BMCC1_HUMAN BNIP2 motif- Q58A63 dspdeidinvDELDTPDEadsfeytghd containing molecule at the C-terminal region 1 - Homo sapiens

UT14C_HUMAN U3 small nucleolar Q5TAP6 pknyplseneDEGDSDGErkhqklleai RNA-associated protein 14 homolog C - Homo sapiens

TBCD9_HUMANTBC1 domain hvlpepssdqDEPDSAFEatqyffedit Q6ZT07 family member 9 - Homo sapiens

DEPD2_HUMAN DEP domain- ssqcssyfhsDEMDSGDElplsvrishd Q70Z35 containing protein 2 - Homo sapiens

NOL8_HUMAN Nucleolar protein 8 - Q76FK4 sgklfdssddDESDSEDDsnrfkikpqf Homo sapiens

K121A_HUMAN Kinesin-like protein Q7Z4S6 eddidggessDESDSESDekanyqadla KIF21A - Homo sapiens

HUWE1_HUMAN E3 ubiquitin- Q7Z6Z7 qeedssgsneDEDDSQDEeeeeeedeed protein ligase HUWE1 - Homo sapiens

NUP93_HUMAN Nuclear pore Q8N1F7 ealqyfyflrDEKDSQGEnmflrcvsel complex protein Nup93 - Homo sapiens

ZC3H8_HUMAN Zinc finger CCCH Q8N5P1 tatdsderidDEIDTEVEetqeekikle domain-containing protein 8 - Homo sapiens BPAEB_HUMAN Bullous Q8WXK8 erectnilegDESDSLTDydivggkesf pemphigoid antigen 1, isoform 7 (Fragment) - Homo sapiens

MYST4_HUMAN Histone Q8WYB5 pshnedhdadDEDDSHMEsaevekeelp acetyltransferase MYST4 - Homo sapiens

TANK_HUMAN TRAF family Q92844 tsvtprglcrDEEDTSFEslskfevkfp member-associated NF-kappa-B activator - Homo sapiens

CLUA1_HUMAN Clusterin- Q96AJ1 ddsdidiqedDESDSELEerrlpkpqta associated protein 1 - Homo sapiens

CC138_HUMAN Coiled-coil domain- Q96M89 fkhvnvncldDELDSFHDlkkqeteeel containing protein 138 - Homo sapiens

MACF4_HUMAN Microtubule-actin Q96PK2 ggsmmmsektDEEDSGREiflscshple cross-linking factor 1, isoform 4 - Homo sapiens

IWS1_HUMAN IWS1 homolog - Q96ST2 sddggatpvqDERDSGSDgeddvneqhs Homo sapiens

Q9C0K7 pwtepecdfpDEKDSYWE

CN093_HUMAN Uncharacterized Q9H972 wndvteelmsDEEDSLNEpgvwvarppr protein C14orf93 precursor - Homo sapiens

CHD8_HUMAN Chromodomain- Q9HCK8 srsvppvkleDEDDSDSEldlsklspss helicase-DNA-binding protein 8 - Homo sapiens

UBN1_HUMAN Ubinuclein - Homo Q9NPG3 qdlidmgygyDESDSFIDnseaydelvp sapiens

PVRL3_HUMAN Poliovirus receptor- Q9NQS3 kesqidvlqqDELDSYPDsvkkenknpv related protein 3 precursor - Homo sapiens

BAZ1A_HUMAN Bromodomain Q9NRL2 seeeeyeveqDEDDSQEEeevslpkrgr adjacent to zinc finger domain protein 1A - Homo sapiens

DDX28_HUMAN Probable ATP- Q9NUL7 sleqlsflvlDEADTLLDesflelvdyi dependent RNA helicase DDX28 - Homo sapiens

CF203_HUMAN Uncharacterized Q9P0P8 kstkkslqkvDEEDSDEEshhdemseqe protein C6or£203 - Homo sapiens

PLCE INHUMAN 1- phosphatidylinositol-4,5-bisphosphate Q9P212 irscnrsletDEEDSPSEgnssrksslk phosphodiesterase epsilon-1 - Homo sapiens

DAPLE_HUMAN Protein Daple - Q9P219 adarsarayrDELDSLREkanrverlel Homo sapiens

IBTK.HUMAN Inhibitor of Bruton Q9P2D0 eefgkllreaDEMDSIHDvtfqvgnrlf tyrosine kinase - Homo sapiens

BAZ2B_HUMAN Bromodomain Q9UIF8 eeeddddkdqDESDSDTEgektsmklnk adjacent to zinc finger domain protein 2B - Homo sapiens BAZ1BHUMAN Bromodomain Q9UIG0 rkkfpdrlaeDEGDSEPEavgqsrgrrq adjacent to zinc finger domain protein IB - Homo sapiens

MAST1_HUMAN Microtubule- Q9Y2H9 sdryhhvnsyDEDDTTEEepveirqfss associated serine/threonine-protein kinase 1 - Homo sapiens [IVL]E.D[ST]..[DE]

MOXD2_HUMAN DBH-like A6NHM9 gnvyfsdqhlVEEDTLKEdgsqdaellg monooxygenase protein 2 precursor - Homo sapiens

LAMA5_HUMAN Laminin subunit 015230 llrfpgyrgcIEMDTLNEevvslynfer alpha-5 precursor - Homo sapiens

MYPT2_HUMAN Protein 060237 earlatltsrVEEDSNRDykklyesalt phosphatase 1 regulatory subunit 12B - Homo sapiens

MAGC1_HUMAN Melanoma- 060732 deytsssdtlLESDSLTDsesliesepl associated antigen CI - Homo sapiens

VAMP4_HUMAN Vesicle-associated 075379 gsvkserrnlLEDDSDEEedfflrgpsg membrane protein 4 - Homo sapiens

SASH1_HUMAN SAM and SH3 094885 ttasstkaqpLEQDSAVDnallltqskr domain-containing protein 1 - Homo sapiens

TRI37_HUMAN Tripartite motif- 094972 peegmssdsdIECDTENEeqeehtsvgg containing protein 37 - Homo sapiens

RIMB1_HUMAN Peripheral-type 095153 clspkcleisIEYDSEDEqeagsggisi benzodiazepine receptor-associated protein 1 - Homo sapiens

CCNE2_HUMAN Gl/S-specific 096020 sievvkkasgLEWDSISEcvdwmvpfVn cyclin-E2 - Homo sapiens

RIBl_HUMANDolichyl- diphosphooligosaccharide—protein P04843 kiilpegaknIEIDSPYEisrapdelhy glycosyltransferase 67 kDa subunit precursor - Homo sapiens

FUMH_HUMAN Fumarate hydratase, P07954 armasqnsfflEYDTFGElkvpndkyyg mitochondrial precursor - Homo sapiens

HSP71_HUMAN Heat shock 70 kDa P08107 rtlssstqasLEIDSLFEgidfytsitr protein 1 - Homo sapiens

ANXA6_HUMAN Annexin A6 • P08133 ekydkslhqalEGDTSGDflkallalcg Homo sapiens

HSP7C_HUMAN Heat shock cognate PI 1142 rtlssstqasIEIDSLYEgidfytsitr 71 kDa protein - Homo sapiens

TPR_HUMAN Nucleoprotein TPR - PI2270 esegisseagLEIDSQQEeepvqasdes Homo sapiens

HSP76_HUMAN Heat shock 70 kDa PI 7066 rtlssstqatLEIDSLFEgvdfytsitr protein 6 - Homo sapiens

pkkmapppkeVEEDSEDEemsedeedds NUCL_HUMAN Nucleolin - Homo P19338 sapiens P20810 aappqekkrkVEKDTMSDqalealsasl ICAL_HUMAN Calpastatin - Homo sapiens

P22064

PUR2_HUMAN Trifunctional purine P22102 vinhklyknrVEFDSAIDlvleefsidi biosynthetic protein adenosine-3 - Homo sapiens

ITA3_HUMAN Integrin alpha-3 P26006 vgkcyvrgndLELDSSDDwqtyhnemcn precursor - Homo sapiens

CTNA2_HUMAN Catenin aIpha-2 - P26232 avlmirtpeeLEDDSDFEqedydvrsrt Homo sapiens

DNMT1_HUMAN DNA (cytosine-5)- P26358 pddvrrrlkdLERDSLTEkecvkeklnl methyltransferase 1 - Homo sapiens

PTPRMJHUMAN Receptor-type P28827 ydhsrvrlqtIEGDTNSDyingnyidgy tyrosine-protein phosphatase mu precursor - Homo sapiens

ARI4A_HUMAN AT-rich interactive P29374 rsvksesditlEVDSIAEesqeglcere domain-containing protein 4A - Homo sapiens

HS71L_HUMAN Heat shock 70 kDa P34931 rtlssstqanLEIDSLYEgidfytsitr protein 1L - Homo sapiens

BMI1_HUMAN Polycomb complex P35226 qrdgltnageLESDSGSDkanspaggip protein BMI-1 - Homo sapiens

MLH1HUMAN DNA mismatch P40692 rhredsdvemVEDDSRKEmtaactprrr repair protein Mlhl - Homo sapiens

AQP2_HUMAN Aquaporin-2 - Homo P41181 lserlavlkgLEPDTDWEerevrrrqsv sapiens

UBE1L_HUMAN Ubiquitin- P41226 wsgpkqcpqpLEFDTNQDthllyvlaaa activating enzyme El homolog - Homo sapiens

CASP3_HUMAN Caspase-3 precursor P42574 acrgteldcglETDSGVDddmachkipv - Homo sapiens

IF2MHUMAN Translation initiation P46199 llntdididsLEADSHLDevwikevitk factor IF-2, mitochondrial precursor - Homo sapiens

HSP77_HUMAN Putative heat shock P48741 rtpssstqatLEIDSLFEgvdfyksitr 70 kDa protein 7 - Homo sapiens

ARRB1_HUMAN Beta-arrestin-1 - P49407 enetpvdtnllELDTNDDdivfedfarq Homo sapiens

P51168 dslrlqpldvIESDSEGDa

UBP14_HUMAN Ubiquitin carboxyl- P54578 mrvlqqklealEDDSVKEtdsssasaat terminal hydrolase 14 - Homo sapiens

HSP72_HUMAN Heat shock-related P54652 rtlssstqasIEIDSLYEgvdfytsitr 70 kDa protein 2 - Homo sapiens

CU063_HUMAN Uncharacterized P58658 reqlvpgsdkVEEDSEDEeeeedpsesd protein C21orf63 precursor - Homo sapiens P78352 qeftecfsaiVEGDSFEEiyhkvkrvie DLG4_HUMAN Disks large homolog 4 - Homo sapiens

ZN200_HUMAN Zinc finger protein P98182 krkmrnllvtIENDTPLEelskyvdisi 200 - Homo sapiens

SATB1_HUMAN DNA-binding Q01826 khfkktkdmmVEMDSLSElsqqganhvn protein SATB1 - Homo sapiens

OCRL_HUMAN Inositol Q01968 revpvtkl idLEEDSFLEkeksllqmvp polyphosphate 5-phosphatase OCRL-1 - Homo sapiens

FKBP4_HUMAN FK506-binding Q02790 saaiescnkaLELDSNNEkglfrrgeah protein 4 - Homo sapiens

SCN1BHUMAN Sodium channel Q07699 alvssacggcVEVDSETEavygmtfkil subunit beta-1 precursor - Homo sapiens

RBL2_HUMAN Retinoblastoma-like Q08999 pasttrrrlfVENDSPSDggtpgrmppq protein 2 - Homo sapiens

BPTF_HUMAN Nucleosome- Q12830 kywflnrrlilEEDTENEnekkiwyyst remodeling factor subunit BPTF - Homo sapiens

COX10_HUMAN Protoheme IX Q12887 rkpnekelieLEPDSVIEdsidvgketk farnesyltransferase, mitochondrial precursor - Homo sapiens

AKAP6_HUMAN A-kinase anchor Q13023 ediecffeacVEGDSDGEepcfssappn protein 6 - Homo sapiens

SEM3B_HUMAN Semaphorin-3B Q13214 karlvcsvpgVEGDTHFDqlqdvfllss precursor - Homo sapiens

TRPC3_HUMAN Short transient Q13507 iaminssyqelEDDSDVEwkfarsklwl receptor potential channel 3 - Homo sapiens

MTR1LJHUMAN Melatonin-related Q13585 agptkpaasqLESDTIADlpdptvvtts receptor - Homo sapiens

LAMC2_HUMAN Laminin subunit Q13753 evegelerkeLEFDTNMDavqmviteaq gamma-2 precursor - Homo sapiens

RB33A_HUMAN Ras-related protein Q14088 qpasaaglasLELDSSLDqyvqirifki Rab-33A - Homo sapiens

BMS1_HUMAN Ribosome biogenesis Q14692 vkgikrrkleLEEDSEMDlpafadsddd protein BMS1 homolog - Homo sapiens

ZN638_HUMAN Zinc finger protein ghsircksknLEDDTLSEckqvsdkavs Q14966 638 - Homo sapiens

PON2_HUMAN Serum Q15165 tnmnltqlkvLELDTLVDnlsidpssgd paraoxonase/arylesterase 2 - Homo sapiens

IF16_HUMAN Gamma-interferon- Q16666 gkkiiiisdyLEYDSLLEvneestvsea inducible protein Ifi-16 - Homo sapiens

PAR14_HUMAN Poly [ADP-ribose] Q460N5 kndvkddrillEFDTLKEmvilagksed polymerase 14 - Homo sapiens Q56UN5 vnasriltndLEFDSVSDhsktltnfsf YSK4_HUMAN SPSl/STE20-related protein kinase YSK4 - Homo sapiens

NOM1JHUMAN Nucleolar MIF4G Q5C9Z4 dagqtlpesdLESDSQDEseeeeegdve domain-containing protein 1 - Homo sapiens

SNPC4_HUMAN snRNA-activating Q5SXM2 gshveisessLESDSEADslpsedldpa protein complex subunit 4 - Homo sapiens

DCST2_HUMAN DC-STAMP Q5T1A1 splsyqgdldLELDSSDEegpqlwlaaa domain-containing protein 2 - Homo sapiens

ZUBR1_HUMAN Zinc finger UBR1- Q5T4S7 icqdcdlvalLEDDSGMEllvnnkiisl type protein 1 - Homo sapiens

RHG21_HUMAN Rho GTPase- Q5T5U3 rselisegrpVETDSESEfpvfptalts activating protein 21 - Homo sapiens

IL23R_HUMAN Interleukin-23 Q5VWK5 nsveeettmlLENDSPSEtipeqtllpd receptor precursor - Homo sapiens

K0240_HUMAN Uncharacterized Q6AI39 kfipdhsegvVETDSILEaavnsile protein KIAA0240 - Homo sapiens

IQEC1_HUMAN IQ motif and Sec7 Q6DN90 nsertqslleLELDSGVEgeapssetgt domain-containing protein 1 - Homo sapiens

FA45B_HUMAN Protein FAM45B - Q6NSW5 dtqlmlgvgllEKDTNGEvlwvwcypst Homo sapiens

FUT10_HUMAN Alpha-(1,3)- Q6P4F1 dsyvrelmtylEVDSYGEclmkdlpqq fiicosyltransferase 10 - Homo sapiens

CO039_HUMAN Uncharacterized Q6ZRI6 gpvmygklprLETDSGLEhslphsvgnq protein C15orf39 - Homo sapiens

NOL8_HUMAN Nucleolar protein 8 - Q76FK4 ddrfrmdsrfLETDSEEEqeevnekkta Homo sapiens

BRAP_HUMAN BRCA1-associated Q7Z569 riywenkivrlEKDTAEEinnmktkfte protein - Homo sapiens

RHG30_HUMAN Rho GTPase- Q7Z6I6 egdqraggyyLEEDTLSEgsgvaslevd activating protein 30 - Homo sapiens

GP123_HUMAN Probable G-protein Q86SQ6 aalvspgpsgVEEDTRREgcvlvgswrs coupled receptor 123 - Homo sapiens

SYT9_HUMAN Synaptotagmin-9 - Q86SS6 llaelcargaLEHDSCQDfiyhlrdrar Homo sapiens

CTGE6_HUMAN Protein cTAGE-6 - Q86UF2 lpmmkdqaavLEEDTTDDdnlelevnsq Homo sapiens

NUDC3_HUMAN NudC domain- Q8IVD9 kepvpvpvqelEIDSTTEldghqevekv containing protein 3 - Homo sapiens

CTGE4JHUMAN Cutaneous T-cell Q8IX94 mmkdqaavLEEDTTDDdnlelkvnsq lymphoma-associated antigen 4 - Homo sapiens

Q8IZQ1 lhgssvtpafVEFDTSLEgfgclflpsl WDFY3_HUMAN WD repeat and FYVE domain-containing protein 3 - 167

Homo sapiens

DSEL_HUMAN Dermatan-sulfate Q8IZU8 tayidipeteLEIDSFVDacewkvsdir epimerase-like protein precursor - Homo sapiens

FMR1N_HUMAN Fragile X mental Q8N0W7 pnsenahgqsLEEDSALEallnfffptt retardation 1 neighbor protein - Homo sapiens

PARP8_HUMAN Poly [ADP-ribose] Q8N3A8 ngeesrqnstVEEDSEGDndseefyygg polymerase 8 - Homo sapiens

CTL3_HUMAN Choline transporter Q8N4M1 lyydytndlsIELDTEREnmkcvlgfai like protein 3 - Homo sapiens

ZN713_HUMAN Zinc finger protein Q8N859 aqleleeewvIERDSLLDthpdgenrpe 713 - Homo sapiens

MARHA_HUMAN Probable E3 Q8NA82 eklkklqeslLEEDSEEEgdlcricqia ubiquitin-protein ligase MARCH 10 - Homo sapiens

EHBP1_HUMAN EH domain-binding Q8NDI1 skasgdendnlEIDTNEEipegfvvggg protein 1 - Homo sapiens

FA45A_HUMAN Protein FAM45A - Q8TCE6 dtqlmlgvgllEKDTNGEvlwvwcypst Homo sapiens

AT8B4_HUMAN Probable Q8TF62 ghvvcekkqqLELDSI VEetitgdy al i phospholipid-transporting ATPase 1M - Homo sapiens

CPLX3_HUMAN Complexin-3 Q8WVH0 velprelakmlEEDTEEEeekasvlgql precursor - Homo sapiens

RIN2_HUMAN Ras and Rab Q8WYP3 ltyviaqcdmLELDTEIEymmelldpsl interactor 2 - Homo sapiens

MY18A_HUMAN Myosin-XVIIIa - Q92614 vtkyqkrknkLEGDSDVDseledrvdgv Homo sapiens

RREB1_HUMAN RAS-responsive Q92766 lpgqpemktqLEQDSIIEallplsmeak element-binding protein 1 - Homo sapiens

PGCB_HUMAN Brevican core Q96GW7 aqapaaladvLEGDSSEDrafrvriagd protein precursor - Homo sapiens

ZFP42_HUMAN Zinc finger protein Q96MM3 virgefsqpiLEGDSLFEsIeylkkgse 42 homolog - Homo sapiens

WDR52_HUMAN WD repeat- Q96MT7 etftkgegsyLEEDSDEErlegslssfq containing protein 52 - Homo sapiens

CCD43_HUMAN Coiled-coil domain- Q96MW1 nvedvfnarkLERDSLRDesqrkkeqdk containing protein 43 - Homo sapiens

CSMD1_HUMAN CUB and sushi Q96PZ7 gflihyesvtLESDSCLDpgipvnghrh domain-containing protein 1 precursor - Homo sapiens

CC123_HUMAN Coiled-coil domain- Q96ST8 rsrsesdvssVEQDSFIEpyattsqlrp containing protein 123, mitochondrial precursor - Homo sapiens

Q9BPX3 cpvvnayatllENDSNPEvrravlscia CND3_HUMAN Condensin complex subunit 3 - Homo sapiens

WDR79_HUMAN WD repeat- yaemvpvlrmVEGDTIYDycwyslmssa Q9BUR4 containing protein 79 - Homo sapiens

SCNM1_HUMAN Sodium channel grgrwvkdenVEFDSDEEeppdlpl Q9BWG6 modifier 1 - Homo sapiens

WNK3_HUMAN Serine/threonine- dneaiefsfhLETDTPEEvayemvksgf Q9BYP7 protein kinase WNK3 - Homo sapiens

SETD2_HUMAN Histone-lysine N- Q9BYW2 eppiiivpesLEADTKQDtisnsleehv methyltransferase SETD2 - Homo sapiens

GRIP2_HUMAN Glutamate receptor- Q9C0E4 tlsspplvcflEPDSPAErcgllqvgdr interacting protein 2 - Homo sapiens

SCND2_HUMAN SCAN domain- Q9GZW5 psvqdlqivkLEEDSHWEqeislqgnyp containing protein 2 - Homo sapiens

EHMT1_HUMAN Histone-lysine N- Q9H9B1 asslhvngesLEMDSDEDdseeleeddg methyltransferase, H3 lysine-9 specific 5 - Homo sapiens

AVEN_HUMAN Cell death regulator Q9NQS1 ggwgagasapVEDDSDAEtygeendeqg Aven - Homo sapiens

RASF1_HUMAN Ras association Q9NS23 gprdlgwepaVERDTNVDepvewetpdl domain-containing protein 1 - Homo sapiens

ABCF3_HUMAN ATP-binding Q9NUQ8 gddtpalqsvLESDSVREdllrrerelt cassette sub-family F member 3 - Homo sapiens

SDA1_HUMAN Protein SDA1 Q9NVU7 apgkcqkrkylEIDSDEEprgellslrd homolog - Homo sapiens

CUED1_HUMAN CUE domain- Q9NWM3 sippeilertLEPDSSDEepppvysppa containing protein 1 - Homo sapiens

SPAT7JHUMAN Spermatogenesis- Q9POW8 ltdretsvnvIEGDSDPEkveisnglcg associated protein 7 - Homo sapiens

Q9UBB5 sraadteemdIEMDSGDE

CS007_HUMAN Zinc finger CCCH Q9UPT8 pgehlfpehpLEPDSFSEggppgrpkpg domain-containing protein C19orf7 - Homo sapiens

ZG14_HUMAN Gastric cancer Q9Y3A4 dsvpdpealrVEVDTFMEaydqkiaeee antigen Zgl4 - Homo sapiens

RRP15_HUMAN RRP15-like protein Q9Y3B9 kdhfysdddalEADSEGDaepcdkenen - Homo sapiens

GRIP1_HUMAN Glutamate receptor- Q9Y3R0 tlsspplisylEADSPAErcgvlqigdr interacting protein 1 - Homo sapiens

VPP2_HUMAN Vacuolar proton feptyeefpsLESDSLLDyscmqrlgak translocating ATPase 116 kDa subunit a isoform 2 - Homo sapiens Q9Y487 DE[ST]D.[DE]

skaeevilaeDETDGEqrhpfdgalr CAC1A_HUMAN Voltage-dependent P/Q-type calcium channel subunit 000555 alpha-lA - Homo sapiens

DDEF2HUMAN Development and 043150 yewrllhedlDESDDDmdeklqpspn differentiation-enhancing factor 2 - Homo sapiens

COBL_HUMAN Protein cordon-bleu 075128 etfrksslgnDETDKEkkkflgffkv - Homo sapiens

CBPDHUMAN Carboxypeptidase D 075976 kksllshefqDETDTEeetlyssk precursor - Homo sapiens

RAG1_HUMANV(D)J P15918 cckplclmlaDESDHEtltailspli recombination-activating protein 1 - Homo sapiens

HNF1A_HUMAN Hepatocyte nuclear P20823 nglgetrgseDETDDDgedftppilk factor 1-alpha - Homo sapiens

P25788 kyakeslkeeDESDDDn

S0X11_HUMAN Transcription factor P35716 efmacspvalDESDPDwcktasghik SOX-11 - Homo sapiens

KCNC1_HUMAN Potassium voltage- P48547 yeeelafwgiDETDVEpccwmtyrqh gated channel subfamily C member 1 - Homo sapiens

INAR2_HUMAN Interferon- P48551 kkkvwdynydDESDSDteaaprtsgg alpha/beta receptor beta chain precursor - Homo sapiens

MRE11_HUMAN Double-strand P49959 tknysevievDESDVEedifpttskt break repair protein MRE11A - Homo sapiens

0D01_HUMAN 2-oxoglutarate dehydrogenase El component, Q02218 stdklgfyglDESDLDkvfhlptttf mitochondrial precursor - Homo sapiens

KCNC4_HUMAN Potassium voltage- Q03721 feeeltfwgiDETDVEpccwmtyrqh gated channel subfamily C member 4 - Homo sapiens

EMAL5_HUMAN Echinoderm Q05BV3 pqesladshsDESDSDlsdvpeldse microtubule-associated protein-like 5 - Homo sapiens

TCOF_HUMAN Treacle protein - Q13428 sssedtssssDETDVEgkpsvkpaqv Homo sapiens

CV019_HUMAN Uncharacterized lfkppedsqdDESDSDaeeeqttkrr Q13769 protein C22orfl9 - Homo sapiens

KCNC3_HUMAN Potassium voltage- Q14003 feeelgfwgiDETDVEaccwmtyrqh gated channel subfamily C member 3 - Homo sapiens

GSE1_HUMAN Genetic suppressor gyyydlddsyDESDEEevrahlrcva Q14687 element 1 - Homo sapiens

NIPBL_HUMAN Nipped-B-like knntaaetedDESDGEdrgggtsgsl Q6KC79 protein - Homo sapiens

Q6P2M8 illcgyppfyDESDPElfsqilrasy KCC1B_HUMAN Calcium/calmodul in-dependent protein kinase type IB - Homo sapiens

LMTK1_HUMAN Serine/threonine- Q6ZMQ8 eeeeedsedsDESDEElrcysvqeps protein kinase LMTK1 - Homo sapiens

NOL8_HUMAN Nucleolar protein 8 - Q76FK4 sgklfdssddDESDSEddsnrfkikp Homo sapiens

KI21A_HUMAN Kinesin-like protein Q7Z4S6 eddidggessDESDSEsdekanyqad KIF21A - Homo sapiens

MYPN_HUMAN Myopalladin - Q86TC9 iefrlertpvDESDDEiqhdeiptgk Homo sapiens

ZFHX4HUMAN Zinc finger Q86UP3 qtslptescsDESDSElsqkledldn homeobox protein 4 - Homo sapiens

CBPC3_HUMAN Cytosolic Q8NEM8 dysdrtisdeDESDEDmfmkfvsedl carboxypeptidase 3 - Homo sapiens

IP04HUMAN Importin-4 - Homo Q8TEX9 dgsssfllfdDESDGEeeeelmdedv sapiens

CLUA1_HUMAN Clusterin- Q96AJ1 ddsdidiqedDESDSEleeirlpkpq associated protein 1 - Homo sapiens

TRI44_HUMAN Tripartite motif- Q96DX7 eeseteeeseDESDEEseedseeeme containing protein 44 - Homo sapiens

DNJA3_HUMAN DnaJ homolog Q96EY1 qqslilsyaeDETDVEgtvngvtlts subfamily A member 3, mitochondrial precursor - Homo sapiens

ZBT45_HUMAN Zinc finger and Q96K62 rgdeddeesdDETDGEdgegggpgeg BTB domain-containing protein 45 - Homo sapiens

TOPK_HUMAN Lymphokine- Q96KB5 nddddedktfDESDFDdeayyaalgt activated killer T-cell-originated protein kinase - Homo sapiens

MACF4JHUMAN Microtubule-actin Q96PK2 qgelmlkkatDETDRDiirepltelk cross-linking factor 1, isoform 4 - Homo sapiens

SETD2_HUMAN Histone-lysine N- Q9BYW2 dysgssessnDESDSEdtdsddssip methyltransferase SETD2 - Homo sapiens

OSBL8_HUMAN Oxysterol-binding Q9BZF1 ksdkendqehDESDNEvmgkseesdt protein-related protein 8 - Homo sapiens

UN93BHUMAN Protein unc-93 Q9H1C4 gyryleednsDESDAEgehgdgaeee homolog Bl - Homo sapiens

ZF106_HUMAN Zinc finger protein kiefqvhaleDESDGEtsdtekhgtk Q9H2Y7 106 homolog - Homo sapiens

ESF1_HUMAN ESF1 homolog - mgtstveitwDETDHEritmlnrkfk Q9H501 Homo sapiens

SG269_HUMAN Tyrosine-protein snmeeeheswDESDEEllameirmrg Q9H792 kinase SgK269 - Homo sapiens

Q9NQZ7 nfvlgrfdheDESDAEatqelaagrr ENTP7_HUMAN Ectonucleoside triphosphate diphosphohydrolase 7 - Homo sapiens

MST4_HUMAN Serine/threonine- Q9P289 krwkaeghsdDESDSEgsdsestsre protein kinase MST4 - Homo sapiens

CPSF2_HUMAN Cleavage and Q9P2I0 qskeadidssDESDIEedidqpsahk polyadenylation specificity factor subunit 2 - Homo sapiens

MIOX_HUMAN Inositol oxygenase - eavdlldglvDESDPDvdfpnsfhaf Q9UGB7 Homo sapiens

BAZ2B_HUMAN Bromodomain Q9UIF8 eeeddddkdqDESDSDtegektsmkl adjacent to zinc finger domain protein 2B - Homo sapiens

BAZ1BHTJMAN Bromodomain Q9UIG0 eesasedsedDESDEEeeeeeeeeee adjacent to zinc finger domain protein IB - Homo sapiens

FBX3_HUMAN F-box only protein 3 Q9UK99 ededddsadmDESDEDdeeerrrrvf - Homo sapiens

DDEF1_HUMAN 130 kDa phosphatidylinositol 4,5-biphosphate- Q9ULH1 yewnlrqeeiDESDDDlddkpspikk dependent ARF1 GTPase-activating protein - Homo sapiens

MACF1HUMAN Microtubule-actin Q9UPN3 qgelmlkkatDETDRDiirepltelk cross-linking factor 1, isoforms 1/2/3/5 - Homo sapiens

MAN1_HUMAN Inner nuclear Q9Y2U8 gskvllgfssDESDVEasprdqaggg membrane protein Manl - Homo sapiens [IVL]E[ST]D.[DE]

RN103_HUMAN RING finger protein 000237 hpiasfqnfpVESDWDedpdlflerl 103 - Homo sapiens

MPP10_HUMAN U3 small nucleolar 000566 ribonucleoprotein protein MPP10 - nlkykdffdpVESDEDitnvhddeld Homo sapiens

HGS_HUMAN Hepatocyte growth 014964 lldkatsqllLETDWEsilqicdlir factor-regulated tyrosine kinase substrate - Homo sapiens

ZN646HUMAN Zinc finger protein gesphgaegnLESDGDclqaesegdk 015015 646 - Homo sapiens

RHG06_HUMAN Rho GTPase- 043182 dlqnevlislLETDPDvvdyllrrka activating protein 6 - Homo sapiens

TXNL1_HUMAN Thioredoxin-like 043396 dnclrkdttfLESDCDeqllitvafn protein 1 - Homo sapiens

ST18_HUMAN Suppression of 060284 mtiklkatgglESDEEirhldeeike tumorigenicity protein 18 - Homo sapiens

A0C2_HUMAN Retina-specific 075106 ihsplgihipLESDMEralswgryql copper amine oxidase precursor - Homo sapiens

075151 fkdsdyvypsLESDEDnpifksrskk PHF2_HUMAN PHD finger protein 2 - Homo sapiens

ITA10_HUMAN Integrin alpha-10 075578 avnmhlgmslLETDGDggfmacaplw precursor - Homo sapiens

ATRN_HUMAN Attractin precursor - 075882 srpfasvnvaLETDEEppdliggsik Homo sapiens

BPAEA_HUMAN Bullous 094833 geedevngnlLETDVDgqvgttqenl pemphigoid antigen 1, isoforms 6/9/10 - Homo sapiens

FRYL_HUMAN Protein furry 094915 atifwiaaslLESDYEyeyllalrll homolog-like - Homo sapiens

FA8_HUMAN Coagulation factor enspsvwqniLESDTEfkkvtplihd P00451 VIII precursor - Homo sapiens

TPM1_HUMAN Tropomyosin alpha- P09493 yeevarklvilESDLEraeeraelse 1 chain - Homo sapiens

MYBB_HUMAN Myb-related protein gsslsealdllESDPDawcdlskfdl PI 0244 B - Homo sapiens

41_HUMAN Protein 4.1 - Homo P11171 sqvseeegkeVESDKEkgeggqkeie sapiens

SKIL_HUMAN Ski-like protein - P12757 ehlddygeapVETDGEhvkrtctsvp Homo sapiens

JAK1_HUMAN Tyrosine-protein kigdfgltkalETDKEyytvkddrds P23458 kinase JAK1 - Homo sapiens

EPHA8_HUMAN Ephrin type-A tcketfhlyyLESDRDlgastqesqf P29322 receptor 8 precursor - Homo sapiens

GNA11HUMAN Guanine P29992 valseydqvlVESDNEnrmeeskalf nucleotide-binding protein subunit alpha-11 - Homo sapiens

L1CAM_HUMAN Neural cell P32004 kdetfgeyrsLESDNEekafgssqps adhesion molecule LI precursor - Homo sapiens

CXA7_HUMAN Gap junction alpha-7 edpmmypemeLESDKEnkeqsqpkpk P36383 protein - Homo sapiens

PTGDS_HUMAN Prostaglandin-H2 P41222 hwgstysvsvVETDYDqyallysqgs D-isomerase precursor - Homo sapiens

SYIC_HUMAN Isoleucyl-tRNA P41252 mearlsalykLESDYEilerfpgayl synthetase, cytoplasmic - Homo sapiens

5HT2B_HUMAN 5- P41595 giaipvpikglETDVDnpnnitcvlt hydroxytryptamine receptor 2B - Homo sapiens

GPR1_HUMAN Probable G-protein nysydldyysLESDLEekvqlgvvhw P46091 coupled receptor 1 - Homo sapiens

ATRX_HUMAN Transcriptional krkpsivtkyVESDDEkplddetvne P46100 regulator ATRX - Homo sapiens

VPS41_HUMAN Vacuolar protein P49754 liyemilhefLESDYEgfatlirewp sorting-associated protein 41 homolog - Homo sapiens GNAQ_HUMAN Guanine nucleotide- P50148 valseydqvlVESDNEnrmeeskalf binding protein G(q) subunit alpha - Homo sapiens

PEX5_HUMAN Peroxisomal P50542 dmeferaksalESDVDfwdklqaele targeting signal 1 receptor - Homo sapiens

P51168 dslrlqpldvIESDSEgda

GBX2HUMAN Homeobox protein P52951 dpkgkeesfsLESDVDyssddnltgq GBX-2 - Homo sapiens

HDAC4HUMAN Histone P56524 qagvqvkqepIES DEEeaepprevep deacetylase 4 - Homo sapiens

MAX_HUMAN Protein max - Homo P61244 msdnddieVESDEEqprfqsaadk sapiens

EVI1_HUMAN Ecotropic virus Q03112 sdlettsgsdLESDIEsdkekfkeng integration site 1 protein homolog - Homo sapiens

SCRN1_HUMAN Secernin-1 - Homo Q12765 ykahewarailESDQEqgrklrstml sapiens

WRNHUMAN Werner syndrome Q14191 ndnendtsyvIESDEDlememlkhls ATP-dependent helicase - Homo sapiens

CASP8_HUMAN Caspase-8 precursor Q14790 qgdnyqkgipVETDSEeqpylemdls - Homo sapiens

CHD4_HUMAN Chromodomain- Q14839 rsssedddldVESDFDdasinsysvs helicase-DNA-binding protein 4 - Homo sapiens

SF3B3_HUMAN Splicing factor 3B Q15393 teqgdifkitLETDEDmvteirlkyf subunit 3 - Homo sapiens

SF3A1_HUMAN Splicing factor 3 Q15459 fgeseevemeVESDEEddkqekaeep subunit 1 - Homo sapiens

DUS6_HUMAN Dual specificity Q16828 IrissdsssdIESDLDrdpnsatdsd protein phosphatase 6 - Homo sapiens

GON4L_HUMAN GON-4-like Q3T8J9 eedetaeeslLESDVEstassprgak protein - Homo sapiens

HYDIN_HUMAN Hydrocephalus- Q4G0P3 pgtfttkrkvIETDPEpahsvleeny inducing protein homolog - Homo sapiens

D0P1_HUMAN Protein dopey-1 - nhtselrsekLETDCEhvqppqwlqt Q5JWR5 Homo sapiens

SNPC4_HUMAN snRNA-activating Q5SXM2 gshveisessLESDSEadslpsedld protein complex subunit 4 - Homo sapiens

TTC18_HUMAN Tetratricopeptide Q5T0N1 yrmfgkqvakLESDMDsetleeqkcq repeat protein 18 - Homo sapiens

FA83B_HUMAN Protein FAM83B - dtlssgtywpVESDVEapnldlgwpy Q5T0W9 Homo sapiens

Q5T5U3 rselisegrpVETDSEsefpvfptal RHG21 HUMAN RhoGTPase- activating protein 21 - Homo sapiens

ZN648_HUMAN Zinc finger protein Q5T619 ahdtqmlsmnLESDDEdggeaekegt 648 - Homo sapiens

DJBP_HUMAN DJ-1-binding protein Q5THR3 nrwsdlsknfLETDNEgngilrrrdi - Homo sapiens

MCTP2_HUMAN Multiple C2 and Q6DN12 sqeeashlhvVETDSEeayaspaerr transmembrane domain-containing protein 2 - Homo sapiens

TTBK2HUMAN Tau-tubulin kinase Q6IQ55 pgeteeksilLESDNEdeklsrgqhc 2 - Homo sapiens

CK063_HUMAN Uncharacterized Q6NUN7 edlhriskdsLESDSEsltqeimchs protein CI lorf63 - Homo sapiens

CH047_HUMAN Uncharacterized Q6P6B1 qriegetgekVETDMEnekvsegaet protein C8orf47 - Homo sapiens

ATAD2_HUMAN ATPase family Q6PL18 ddsqnaidhklESDTEetqdtsvdhn AAA domain-containing protein 2 - Homo sapiens

UBP34HUMAN Ubiquitin carboxyl- Q70CQ2 ttlisafrilLESDEDrllvvfiirgl terminal hydrolase 34 - Homo sapiens

NOL8_HUMAN Nucleolar protein 8 - Q76FK4 ddrfrmdsrfLETDSEeeqeevnekk Homo sapiens

CEP68_HUMAN Centrosomal protein Q76N32 tesrwkseeeVESDDEylalparltq of 68 kDa - Homo sapiens

ARMC9_HUMAN LisH domain- Q7Z3E5 InseelpdgvLESDDDededdeedhd containing protein ARMC9 - Homo sapiens

CSMD2_HUMAN CUB and sushi Q7Z408 fgfqrldlrlLESDPEsigrhfasns domain-containing protein 2 - Homo sapiens

DNJB7_HUMAN DnaJ homolog Q7Z6W7 ngrnintkkilESDQEreaedngelt subfamily B member 7 - Homo sapiens

CLC14_HUMAN C-type lectin Q86T13 prkesmgppgLESDPEpaalgsssah domain family 14 member A precursor - Homo sapiens

ALPK2_HUMAN Alpha-protein Q86TB3 nllgtehvflLESDDEemefgehclg kinase 2 - Homo sapiens

PEX5R_HUMAN PEX5-related Q8IYB4 eeeferakaaVESDTEfwdkmqaewe protein - Homo sapiens

FANCMHUMAN Fanconi anemia Q8IYD8 aplpaaaeaqLESDDDvllvaayeae group M protein - Homo sapiens

THNS1_HUMAN Threonine sqrengwavgVESDFDfcqtaikrif Q8IYQ7 synthase-like 1 - Homo sapiens

PELP1_HUMAN Proline-, glutamic Q8IZL8 keeasdveisLESDSDdsvvivpegl acid- and leucine-rich protein 1 - Homo sapiens

Q8N2G8 avelldvflgLETDGEelagaiaagn GHDC_HUMAN GH3 domain- containing protein precursor - Homo sapiens

CA052HUMAN Uncharacterized arllpegeetLESDDEkdehtskkrk Q8N6N3 protein Clorf52 - Homo sapiens

PSD4_HUMAN PH and SEC7 Q8NDX1 wtldasqsslLETDGEqpsslkkkea domain-containing protein 4 - Homo sapiens

NAV1_HUMAN Neuron navigator 1 - flvrylrrklVESDSDinankeellr Q8NEY1 Homo sapiens

NEK11_HUMAN Serine/threonine- yyadafdsycVESDEEeeeialerpe Q8NG66 protein kinase Nekl 1 - Homo sapiens

RPGF6_HUMAN Rap guanine Q8TEU7 kvkqylssldVETDEEkfqmmsIqwe nucleotide exchange factor 6 - Homo sapiens

JPH3_HUMAN Junctophilin-3 - Q8WXH2 sppqksIpvaLESDEEngdelksstg Homo sapiens

CDKL2_HUMAN Cyclin-dependent Q92772 tgrivaikkfLESDDDkmvkkiamre kinase-like 2 - Homo sapiens

JKIP2_HUMAN Janus kinase and Q96AA8 eaalhqkmmeLESDMEqfckikgyle microtubule-interacting protein 2 - Homo sapiens

VP13A_HUMAN Vacuolar protein Q96RL7 kmkkkakmaiVESDPEeenykvpeyk sorting-associated protein 13A - Homo sapiens

RSF1_HUMAN Remodeling and gkpsrkrlhrlETDEEescdnahgda Q96T23 spacing factor 1 - Homo sapiens

SH3G1_HUMAN SH3-containing Q99961 evaetsmhnlLETDIEqvsqlsalvd GRB2-like protein 1 - Homo sapiens

LPIN3_HUMAN Lipin-3 - Homo Q9BQK8 dsgeaffvqeLESDDEhvppglctsp sapiens

ORAI3_HUMAN Protein orai-3 - Q9BRQ5 famvamvevqLESDHEyppgllvafs Homo sapiens

SYTM_HUMAN Threonyl-tRNA Q9BW92 ngepydlerpLETDSDlrfltfdspe synthetase, mitochondrial precursor - Homo sapiens

ZN416HUMAN Zinc finger protein Q9BWM5 dqkhhsaekpLESDMDkasfvqcclf 416 - Homo sapiens

SCAPE_HUMAN S phase cyclin A- Q9BY12 ieaeenndinlETDNDsdfsasmgsg associated protein in the ER - Homo sapiens

WNK3_HUMAN Serine/threonine- Q9BYP7 epttlqpttvLESDGErppklefadn protein kinase WNK3 - Homo sapiens

SETD2_HUMAN Histone-lysine N- Q9BYW2 rgplkkrrqelESDSEsdgelqdrkk methyltransferase SETD2 - Homo sapiens

CRNL1_HUMAN Crooked neck-like Q9BZJ0 ydawfdylrlVESDAEaeavrevyer protein 1 - Homo sapiens

Q9C0OO mahfeemgmcVETDMEllvctfcikf NALP1_HUMAN NACHT, LRR and PYD domains-containing protein 1 - Homo sapiens

ZN407_HUMAN Zinc finger protein qeaqgehmdlVESDGEisqiivteel Q9C0GO 407 (Fragment) - Homo sapiens

CA128_HUMAN UPF0424 protein weertdrskfVESDADeellfhipft Q9GZP4 Clorfl28 - Homo sapiens

sqkshtitrpLESDEDekaftissnp PEG3_HUMAN Paternally-expressed Q9GZU2 gene 3 protein - Homo sapiens

ZCPW1_HUMAN Zinc finger CW- Q9H0M4 qygypwwpgmlESDPDlgeyflftsh type PWWP domain protein 1 - Homo sapiens

ROB02_HUMAN Roundabout Q9HCK4 tiewykdgerVETDKDdprshrmllp homolog 2 precursor - Homo sapiens

KCNK9_HUMAN Potassium channel Q9NPC2 llvgaavfdaLESDHEmreeeklkae subfamily K member 9 - Homo sapiens

PRDM5_HUMAN PR domain zinc Q9NQX1 enifylavedlETDTElligyldsdm finger protein 5 - Homo sapiens

BAZ1AJHUMAN Bromodomain Q9NRL2 rrlssrqrpsLESDEDvedsmggedd adjacent to zinc finger domain protein 1A - Homo sapiens

MA2C1_HUMAN Alpha- Q9NTJ4 lssprqlfsaLESDSEqlctwvgelf mannosidase 2C1 - Homo sapiens

MDN1_HUMAN Midasin - Homo Q9NU22 vlsepcrsslVESDKEeqpdflprpt sapiens

RPC2_HUMAN DNA-directed RNA Q9NW08 piviifkamgVESDQEivqmigteeh polymerase III subunit RPC2 - Homo sapiens

WDR70JHUMAN WD repeat- Q9NW82 ktqpktmfaqVESDDEeaknepewkk containing protein 70 - Homo sapiens

PLCE1_HUMAN 1- phosphatidylinositol-4,5-bisphosphate Q9P212 fqvirscnrsLETDEEdspsegnssr phosphodiesterase epsilon-1 - Homo sapiens

RHG23_HUMAN Rho GTPase- Q9P227 adderselshVETDTEgaagagpggr activating protein 23 - Homo sapiens

CHD7_HUMAN Chromodomain- Q9P2D1 kedelmefsdLESDSEekpcakprrp helicase-DNA-binding protein 7 - Homo sapiens

FRM4A_HUMAN FERM domain- Q9P2Q2 ggatpvvvrsLESDQEchysvkaqfk containing protein 4A - Homo sapiens

PHF8_HUMAN PHD finger protein 8 fkdaeyiypsLESDDDdpalksrpkk Q9UPP1 - Homo sapiens

HIF3A_HUMAN Hypoxia-inducible Q9Y2N7 lftsgkdteaVETDLDiaqdadaldl factor 3 alpha - Homo sapiens

WNK2_HUMAN Serine/threonine- gpqrflrrsvVESDQEeppgleaaea Q9Y3S1 protein kinase WNK.2 - Homo sapiens

Q9Y471 fkdynlvvrmlETDEDfnpfpggydy CMAH_HUMAN Cytidine monophosphate-N-acetylneuraminic 177

acid hydroxylase-like protein - Homo sapiens

DMXL1_HUMAN DmX-like protein Q9Y485 tqllmtdthmLETDEEntkprvidls 1 - Homo sapiens

UBP15_HUMAN Ubiquitin carboxyl- Q9Y4E8 kgasaatgipLESDEDsndndndien terminal hydrolase 15 - Homo sapiens

ROB01_HUMAN Roundabout Q9Y6N7 tiewykggerVETDKDdprshrmllp homolog 1 precursor - Homo sapiens

PCLO_HUMAN Protein piccolo - Q9Y6V0 rsscseyspsIESDPEgfeispekii Homo sapiens

Table A-2. Peptides containing an overlapping CK2/caspase recognition sequence phosphorylated by GST-CK2a on peptide arrays. Relative [y- P] incorporation determined using ImageQuant TL software. Fold above negative GI identification Positive Peptide AVERAGE control number (controls) 970221.3533 113.67 24308177 968820.6633 113.50 50959085 50959102 50959110 50959115 839045.7433 98.30 94681063 785217.93 91.99 FSDFDEKTDDEDF TOP2A_HUMAN 765362.4267 89.67 46048234 700945.1233 82.12 24234690 5031923 682118.72 79.92 14670392 623777.71 73.08 148596992 607580.98 71.18 46048234 600140.86 70.31 34101288 593054.2633 69.48 124301196 586223.86 68.68 7706029 561968.49 65.84 157885806 560216.6167 65.63 37620206 559904.54 65.60 154426272 530914.5333 62.20 21361639 529373.0267 62.02 FSDFDEKTDDEDF TOP2A_HUMAN 524500.0733 61.45 45593144 520029.5533 60.93 58000461 513595.7867 60.17 114326455 508526.3733 59.58 14249464 503276.6567 58.96 134304844 494378.2 57.92 109633026 109633028 15011880 483468.85 56.64 46094081 482798.71 56.56 46048234 480185.3667 56.26 30410779 465773.8567 54.57 IDLFGSDNEEEDK EF-1 delta 464846.9633 54.46 KIEDVGSDEEDDS HS90B_HUMAN 463105.3367 54.26 89059898 89060894 450880.46 52.82 156151430 442614.0267 51.86 150456444 441611.64 51.74 21361837 439348.0333 51.47 KIEDVGSDEEDDS HS90BHUMAN 437047.0833 51.20 38569484 435885.22 51.07 51702222 434066.4133 50.85 IDLFGSDNEEEDK EF-1 delta 428024.53 50.15 42544207 420329.0733 49.24 119874201 404803.89 47.43 4758956 403493.5067 47.27 21361800 383023.85 44.87 40217808 378307.63 44.32 30410779 376564.5833 44.12 22094143 374250.5033 43.85 88963230 353976.38 41.47 124301196 335274.3367 39.28 153085395 331769.4267 38.87 154800457 331266.3333 38.81 113418444 113418942 113419444 27485460 45580709 89034947 327592.05 38.38 19923796 313663.1133 36.75 4759274 307995.4033 36.08 12293719189066852 307418.2 36.02 23110939 4506183 290431.8933 34.03 116812624 283136.74 33.17 38788333 281157.1433 32.94 50959085 50959102 50959110 50959115 280329.0367 32.84 28416946 280194.7267 32.83 94681063 276503.14 32.39 16507198 274096.28 32.11 62460637 272236.3033 31.89 2351039123510393 269887.16 31.62 5174715 268105.67 31.41 110735439 89062970 267538.7767 31.34 150170670 150378539 264985.0667 31.04 22202611 262809.6833 30.79 66912176 261600.7667 30.65 15812186 261386.7733 30.62 89050226 89056996 252437.7833 29.57 30410779 246920.41 28.93 153792294 245347.53 28.74 5031825 243056.3567 28.48 92859678 242538.1533 28.42 22202611 241052.21 28.24 51599156 237982.21 27.88 148368962 223697.7433 26.21 157364943 157364945 157364947 223359.96 26.17 46048234 222173.8067 26.03 9966841 221604.44 25.96 13435145 52345626 217887.63 25.53 50657352 217108.18 25.44 4502249 213266.4767 24.99 50345879 212059.69 24.84 61097912 211441.8233 24.77 44955926 205948.4867 24.13 7706671 205893.34 24.12 15619008 66346672 66346674 66346676 205609.6233 24.09 18490991 194108.9767 22.74 13435145 52345626 191940.5967 22.49 146262005 56847634 190880.0933 22.36 55770846 185171.8833 21.69 46488937 184885.2267 21.66 RRREEETEEEEEE CK2 positive PEPTI1 182449.4167 21.38 113423509 179895.02 21.08 21493045 173759.7067 20.36 151301080 173604.5333 20.34 27734911 172308.05 20.19 5032087 53831995 171511.8833 20.09 34485720 171038.47 20.04 50845416 50845418 170832.3033 20.01 122939165 168399.4633 19.73 31317305 162461.6933 19.03 92859678 162114.8267 18.99 126090663 160901.88 18.85 154937340 154937342 154475.4633 18.10 RRREEETEEEEEE CK2 positive PEPTI 151861.5033 17.79 24497618 148798.7867 17.43 61676188 148728.45 17.42 KPATPAEDDEDDD EF-1 delta 148106.2133 17.35 22129784 147822.59 17.32 32967603 32967605 145433.3833 17.04 8922301 142586.08 16.71 38569484 135295.1467 15.85 55956788 132753.7467 15.55 14149720 129739.1167 15.20 11968023 127963.86 14.99 7706135 127825.0067 14.98 4885431 5123454 127172.2567 14.90 4507687 126548.9233 14.83 14210490 120251.94 14.09 8922976 115082.8167 13.48 50845416 50845418 114742.8067 13.44 113418582 113418654 113419608 113419630 113419664 114548.11 13.42 82830424 82830428 112312.27 13.16 8923657 109751.0667 12.86 113430242 20336205 20336209 109347.7967 12.81 33354285 108558.3967 12.72 9966821 107470.8967 12.59 118918423 107145.81 12.55 46488937 12.42 4758996 180

105683.4367 12.38 39930610 4506805 104910.4533 12.29 114199473 114199475 104122.85 12.20 3837293138372935 99135.5 11.61 34577118 98715.13333 11.57 113421353 113421886 97965.53333 11.48 54112403 97356.84 11.41 28144897 28144899 97074.48333 11.37 33188443 33188445 96277.68667 11.28 153791839 95884.52 11.23 113419592 95850.55 11.23 20149588 25777684 94065.76667 11.02 63252863 93386.29333 10.94 15147333 52487176 91397.96667 10.71 4557529 90333.11 10.58 54633319 90124.24667 10.56 54112121 90078.91 10.55 19923796 89054.00667 10.43 41349476 87698.98 10.27 18079218 51243032 84809.54333 9.94 20070344 84751.75333 9.93 32490561 47080103 84339.63333 9.88 40217799 40217801 82927.32667 9.72 46852164 82267.01667 9.64 42764683 81163.05333 9.51 109240536 80457.91667 9.43 121256621 4758172 80214.79667 9.40 94721239 94721241 80183.76 9.39 QACRGTELDCGIE 79634.02 9.33 54792778 79388.25333 9.30 QACRGTELDCGIE 79194.32 9.28 40255087 79159.87667 9.27 113424745 113425014 78805.55667 9.23 134133288 78120.05 9.15 40254431 77872.30667 9.12 117168250 77825.37333 9.12 4505117 75277.2 8.82 10140845 55770892 75239.76333 8.81 24307961 75192.89 8.81 18093112 73915.06667 8.66 113424241 113424453 27597085 63252896 63252898 63252900 63252902 63252904 73822.29 8.65 63252906 73701.86333 8.63 74959747 73499.48333 8.61 21704283 73180.07 8.57 4506675 72700.81 8.52 13128998 72157.42333 8.45 19743569 72102.12667 8.45 50345879 72067.27667 8.44 YDELQTDGNRSSH BID_HUMAN 71178.46333 8.34 24497460 70409.27333 8.25 54792088 69768.83333 8.17 4506929 67616.73667 7.92 22748651 67518.94 7.91 28269707 67068.07333 7.86 47578105 47578107 67051.04 7.86 4505293 66769.59667 7.82 109637791 65652.34667 7.69 55741661 64228.8 7.52 21704261 21704265 21704267 21704269 37577142 63353.89333 7.42 66529294 66529396 62691.92 7.34 124256489 62316.78667 7.30 148612853 61470.57667 7.20 7382480 95091875 95091881 95091988 95092150 61447.42333 7.20 7662168 61146.60667 7.16 5032005 60921.89667 7.14 62899075 62899077 60562.64333 7.10 31581524 59677.91 6.99 113430631 58530876 58354.76667 6.84 151101337 58316.96 6.83 21553335 57589.58333 6.75 32261320 7706577 57268.18 6.71 8922396 57102.18 6.69 31543831 56819.8 6.66 32698700 56647.17 6.64 YDELQTDGNRSSH BID HUMAN 55662.24333 6.52 7706607 55376.54667 6.49 92110053 55332.22667 6.48 22538488 22538490 54862.57667 6.43 MSSSEEVSWISWF CK2 beta 53987.89 6.33 155030232 53950.45333 6.32 71725345 53852.56667 6.31 113423509 53844.44333 6.31 11559923 14702180 89026104 89026106 53771.87 6.30 31317305 53397.34 6.26 31377846 53299.71667 6.24 153792148 53137.03667 6.23 21361292 52996.13333 6.21 34577047 34577049 52842.62333 6.19 13325072 52753.95667 6.18 113417060 24432009 52600.72 6.16 74271888 52375.98 6.14 21450861 52297.43667 6.13 4758528 52038.66 6.10 5146081188957916 51680.60667 6.05 113425649 117168248 109637753 109637757 109637759 109637761 109637763 51499.86333 6.03 109637765 109637767 109637769 109637771 27765085 51305.72333 6.01 MSSSEEVSWISWF CK2 beta 50994.86333 5.97 33438600 5.84 20357504 49791.57667 5.83 113411710 113412646 49710.35 5.82 38570107 38570109 48522.31 5.68 41281437 48508.13333 5.68 4507161 48293.84 5.66 119874201 48291.38 5.66 122939172 69122473 48244.77333 5.65 53729337 53729339 48056.08667 5.63 24415404 47603.74 5.58 14149627 47184.30333 5.53 89179323 46659.44333 5.47 30840980 46559.45 5.45 21704263 46559.36333 5.45 4505569 46141.93333 5.41 4506791 46131.82 5.40 41281441 45916.46 5.38 4557841 45718.02667 5.36 38569398 45421.4 5.32 22749257 45002.38333 5.27 38788260 38788274 44767.98333 5.24 57164977 57164979 44452.27667 5.21 81158222 44371.26 5.20 113426861 44342.09333 5.20 23397642 43833.12333 5.14 4503647 43822.96333 5.13 19743875 43343.02 5.08 4503729 43079.43667 5.05 MEEDSYDSFGEPS PSN2JHUMAN 42148.02667 4.94 116063562 41617.83333 4.88 51871374 41481.47333 4.86 14670392 40983.50667 4.80 20631958 20631967 20631973 4757838 40908.66 4.79 40805106 40825.34 4.78 45237195 40794.99667 4.78 47578115 40387.97 4.73 50083277 40377.68 4.73 14719829 14719833 14719835 74271814 7662386 40279.57 4.72 58331268 39958.17333 4.68 82659109 39861.9 4.67 11386199 39741.33333 4.66 60498984 39685.15333 4.65 CVLLSHGEEGIIF 39637.41667 4.64 24308023 39327.31333 4.61 28376627 39161.79667 4.59 57164975 39133.14667 4.58 115511049 40254462 38986.24 4.57 32967603 32967605 38813.64667 4.55 94721344 38772.55 4.54 31317272 38729.37333 4.54 4503915 38113.97333 4.47 KPATPAEDDEDDD EF-1 delta 37953.08667 4.45 113414960 122937249 37485.63333 4.39 32455273 37274.95667 4.37 148596949 36788.4 4.31 21359945 35962.81 4.21 149999378 149999380 35839.08333 4.20 16357477 35766.64333 4.19 153791300 35626.03667 4.17 21264602 35414.97333 4.15 88759331 35057.01667 4.11 66773344 34499.94333 4.04 118572601 118572603 34476.63667 4.04 42741679 34138.24333 4.00 103472122 113423396 113423398 113423813 113423815 34026.36 3.99 113424943 148762940 89037763 33929.64333 3.98 50083293 33819.84667 3.96 113419876 13027388 89026573 33742.26333 3.95 113415112 19743806 4506569 61888896 33709.02 3.95 33859678 33702.67667 3.95 21626468 62526045 33560.07333 3.93 124256496 33229.52667 3.89 40538726 40788001 32924.63667 3.86 48976051 32854.11667 3.85 31563534 32796.32667 3.84 4557707 32628.13333 3.82 33667030 32580.68333 3.82 4557757 32376.63667 3.79 11345486 32142.97667 3.77 102469034 31878.28333 3.73 148536844 148536846 31430.68667 3.68 MEEDSYDSFGEPS PSN2_HUMAN 31253.96 3.66 14790115 14790119 31147.37667 3.65 27883842 21217561 21217563 24497458 24497462 24497464 31137.23333 3.65 482678688758576 30874.46 3.62 113429619 25092725 30749.31 3.60 22748795 29739.27 3.48 94536848 94536850 29532.88667 3.46 148228829 36951012 28712.74 3.36 45120115 28668.26333 3.36 4502179 28073.27 3.29 CVLLSHGEEGIIF 27859.73667 3.26 113421225 27841.51 3.26 33239445 83367072 27794.73333 3.26 71773329 71773415 26833.65667 3.14 122056474 122056476 15718704 15718706 15718708 26551.88667 3.11 4504747 6006011 26405.88333 3.09 18466802 26300.02333 3.08 89037239 26292.5 3.08 4885599 26249.79 3.08 17921982 184

25932.93 3.04 24234686 5729877 25833.82333 3.03 19923987 25805.09667 3.02 4503351 25163.98667 2.95 23065535 9042131190421316

7752.05 ive control 185

APPENDIX B

B.l Materials and Methods

B.l.l Detection of inhibitor-dependent phosphorylation changes using functional proteomics

Proteins were purified using triazol extraction from [y- P]-labeled HeLa cells treated with DMSO or 25 uM DMAT for 18 hrs. Proteins were run on IPG strips pH 4-7, 7 cm strips followed by 2D gel electrophoresis and [g-32P] incorporation was detected using autoradiography or Pro-Q diamond stain (Invitrogen). Total protein levels were detected using Sypro-Ruby protein stain (Invitrogen). Following staining of 2D gels, spots were picked from gels manually or by the Ettan Spot Picker (Amersham) and suspended in 50% methanol and 5% acetic acid. Trypsin digestion was performed on excised spots using the MassPREP automated digestor (Waters). Peptides were lyophilized, suspended in 30% ACN/0.1% TFA mixed with a-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN/50% 25 mM ammonium citrate/0.1% TFA and analyzed by MALDI MS and/or MS/MS on the 4700 proteomics analysis MALDI (TOF:TOF) instrument (Applied Biosystems). MS and/or MS/MS analysis was carried out with an m/z range of 800-4000 Da and mass tolerance of 50 ppm with a resolution of approximately 15,000. Peptide fingerprinting was evaluated using GPS Explore Workstation version 3.0 series (Applied Biosystems) in conjunction with MASCOT using an internal calibration with a min S/N threshold of 20, peptide mass exclusion tolerance of 50 ppm, 5 minimum peaks to match and a maximum outlier error of 15 ppm with no more than 1 missed cleavage. Mass references consisted of des-Argl-Bradykinin (904.468 Da), Angiotensin 1 (1296.685 Da), Glul-Fibrinopeptid (1570.677 Da), ACTH (1-17) (2093.087 Da), ACTH (18-39) (2465.199 Da), while the mass exclusion list consisted of (842.5099, 870.509, and 2211.1096 Da). Peptides were searched against the human Swiss-Prot database (release 54.0 July 24 2007, 276,256 sequence entries) with oxidation as a variable modification (M). 186

B.1.2 Caspase-3 cleavage of CK2

Cleavage of CK2 by caspase-3 was assayed via incubation of 1 mg of GST-CK2a with 1 uL of purified active caspase-3 (>7000 U/mL) incubated in caspase 3 assay buffer (50 mM HEPES, (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 mM DTT) overnight at 30 °C with agitation. Following caspase incubation, CK2 kinase activity was assayed in a kinase assay using a-casein as a substrate. Cleaved GST-CK2a was determined by Western blot analysis using anti-CK2ct N-terminal antibody. Cleavage of CK2 in cell lysates was performed via the incubation of 20 mg of HeLa cell lysate over-expressing CK2a-HA/CK2(3 with 1 uL of purified caspase-3 (>7000 U/mL) overnight in caspase-3 assay buffer at 30°C with agitation. Cleavage of CK2 was determined by Western blot analysis utilizing the anti-CK2a N-terminal antibody. The global caspase inhibitor z-VAD (calbiochem) was used as a control.

B.1.3 Western Analysis

HeLa cells were harvested from 10 and 15 cm plates (Falcon) in lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.0%

'inf. IQI the C-terminal synthetic peptide a " and a polyclonal anti-CK2a antiserum directed against an N-terminal synthetic peptide a2-19. Membranes were washed with PBST or TBST then incubated in appropriate secondary antibodies, including: (BioRAD) HRP- GAR and HRP-Biotin (Jackson ImmunoResearch) (1:10000 for HA 3F10 and cMyc 9E10). Following secondary antibody incubation, membranes were washed with PBST 187 or TBST and visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia). X-ray film (Kodak) was developed and converted to a digital image using CanoScan N650U/N656U scanner. Images were visualized in Adobe Photoshop CS.

B. 1.4 Cell culture and transfections

HeLa cells were cultured in DMSO (Gibco) containing 10% fetal bovine serum (Gibco), penicillin (100 U/mL) and streptomyocin (100 mg/mL) (Gibco) on 10 cm plates (Falcon). HeLa cells were transfected with either CK2o>HA/HA-CK2p\ CK2o>HA (K68M)/HA-CK2p in addition to pro-caspase-3-myc (Addgene).

B.1.5 Generation ofpro-caspase-3 CK2 phosphorylation mutants

Catalytically-inactive His-tagged pro-caspase-3 (ATCC) was engineered using a QuickChange II Site-Directed Mutagenesis Kit (Stratagene). Human pro-caspase-3 pET23b was PCR amplified to make the (CI63A) mutation using the primer 5- TTCATTATTCAGGCCGCCCGTGGTACAGAA-3'. Phosphorylation mutants (T174A), (SI76A) and (T174A SI76A) of pro-caspase-3 were generated using the following s 5 primers -GACTGTGGCATTGAGGCAGACAGTGGTGGTGAT-3', "GGCATTGAGACAGACGCTGGTGGTGATGAC-'',

S'-GACTGTGGCATTGAGGCAGACGCTGGTGTTGATGATGAC-3', respectively. 188

[Y-*P]

Pro-Q-Diamond f "

Sypro-Ruby

DMSO DMAT

Figure B-l. Identification of CK2 inhibitor-dependent changes in phosphorylation using functional proteomics. HeLa cells were labeled with [g-32P] and treated either with DMSO or 25 \iM DMAT for 18 hrs. Cells were harvested and subjected to 2D gel electrophoresis, followed by Pro-Q diamond and Sypro-Ruby staining. Relative [y-32P] was detected using autoradiography. Changes in phosphorylation are highlighted with red circles. 189

Table B-l. Putative CK2 inhibitor-biomarkers identified using functional proteomics. Proteins identified using mass spectrometry (ESI and/or MALDI). Detection of phosphorylated proteins included 3 P-labeling of cells, Pro-Q diamond staining and the use of phospho-antibodies (MPM-2, phospho-Thr/Pro or P-ser).

Putative Target Detection Protein Name Phosida MS analysis Phospho-site In vivo method Nm23Hl IIHGSDSVESAE 2P, Pro-Q ESI and ERTFIAIKPDGV MALDI

CPa FDDRVSDEEKVR ESI and 2P, Pro-Q MALDI

PAK1 PPVSEDEDDDDD 32 ESI and X P, Pro-Q MALDI

EF-1 delta KPATPAEDDED 2P, P-ser, MPM2, X X MALDI DIDLFGSDNEEE P-Thr/pro

d(UTPase) MPCSEETPAISP 32 ESI and P, Pro-Q MALDI

14-3-3 epsilon DTLSEESYKDST 32P, Pro-Q ESI

HSP60 SKPVTTPEEIAQ X 32P, P-ser MALDI KIMQSSSEVGYD Nucleophosmin ESEDEEEED X X 32P, P-ser MALDI 190

CK2cx-HA HA-CK2a' IP: anti-HA WB: Myc Mycj3-Pi HA Myc|3

Figure B-2. CK2 gatekeeper mutants express and retain kinase activity in cells. HeLa cells were transfected with either CK2a-HA (Fl 13G) or CK2a'-HA (Fl 14A) along with Myc-CK2p\ followed by immunoprecpitation for CK2a-HA (F113G) or CK2a'-HA (F114A) using anti-HA antibodies. Phosphorylated Myc-CK2|3 in complex with CK2a- HA (F113A) or CK2a'-HA (F114A) was assessed using anti-Myc antibodies in Western blot analysis. 191

Cleaved caspase-3 (17kDa) #

pro-caspase-3-myc + pro-caspase-3 TSA-myc + etoposide +

Figure B-3. Mutation of the CK2 phosphorylation sites in pro-caspase-3 generates a non-cleavable form of pro-caspase-3. HeLa cells were transfected with either wild-type pro-caspase-3-my c or pro-caspase-3-myc mutants (T174A/S176A) and treated with DMSO or 25 mM etoposide for 24 hrs. Cleavage of pro-caspase-3-myc was detected via Western blot analysis using anti-caspase-3 antibodies. 192

B IB: anti- CK2a [y«p]

vmmim^mmmmmmMmmmwiimiismmmmmmm

GST-CK2a *4* •*M <*& <*m •*#•

Osgradation 4§ Cleaved products CK2a a-caseina»r

«mmrn^»9mm CK2ct + + + + + Casp3 + - - + - Casp 3 C163A - + - - - Z-VAD - - - + + GST-CK2a + a-casein + + + + + Caspase3 +

IB: anti- CK2ct

Human ELLVDYQMYDYSLDHWSLGCMLASMIFRKEPFFBGHDNYDQLVRIAKVLG 250 CK2ct-HA Mouse ELLVOYOMYDYSLMWSLCCMLMMIFWtEJFFaGHDNYDOLVRIAKVLG 250 Dresephila IUVDYQHyDYSUWini«CHLMNinwSPFF«eiDllYD«,VItUXVL6 24a CK2a MbrafUh lUVDYQIffDYSLDMHSLGCMLASMIFOItnrFaOQDNYDOLVKUUCVLG 250 *************************** I ******* I **************

Cleaved CK2a (28 kDa) Putative Kinase caspase 3 motif site V V CK2a + + + + D (16.5 kDa) 240 Caspase 3 - - + + (28.5 kDa) C163A Caspase 3 - + - - Z-VAD _ . . +

Figure B-4. Cleavage of CK2a by caspase-3 in vitro. (A) To investigate the caspase mediated cleavage of CK2, GST-CK2a was incubated with active caspase-3, and cleavage was assayed using Western blot probing membranes with anti-CK2cc N-terminal antibody. Generation of an approximately (28 kDa) N-terminal proteolytic fragment was observed. (B) The effect of cleavage on kinase activity was determined where CK2 was pre-incubated with active caspase-3 followed by a CK2 kinase assay using a-casein as a substrate. (C) To test whether CK2 could be cleaved by caspase-3 amongst all other cellular proteins, purified active caspase 3 was incubated with HeLa lysates expressing CK2a-HA, and cleavage was determined via Western blot using the anti-CK2a N- terminal antibody. Generation of a z-VAD sensitive (28 kDa) N-terminal proteolytic fragment was observed. (D) An analysis of the CK2a protein sequence revealed a putative caspase 3-cleavage site at Asp 240 (DNYD). CURRICULUM VITAE

NAME: James Stuart Duncan DATE OF BIRTH CURRENT DATE July 2008

EDUCATION University of Western Ontario PhD Biochemistry 2003-present University of Western Ontario B.Sc. Hon. Biochemistry 1998-2003

HONORS & AWARDS

2007-present Canadian Graduate Scholarship Doctoral Award (CIHR) (Valued at $35,000 for 12 months) 2007-present Ontario Graduate Scholarship-declined (Valued at $15,000 for 12 months) 2006-2007 Ontario Graduate Scholarship In Science and Technology (Valued at $15,000 for 12 months) 2005-present CIHR-UWO Strategic Training Initiative in Cancer Research and Technology Transfer (Valued at $25,000 for 12 months) 2004-2005 Ontario Graduate Scholarship (Valued at $15,000 for 12 months) 2003-2004 Special University Scholarship (Valued at $4,500 for 12 months) 2002-2003 Excellence in Biochemistry of DNA/RNA (BCH410a) (Valued at $100) 2002 Biochemistry Studentship Award (Valued at $4,000 for summer) 194

1998-2002 Dow Chemical Higher Education Assistance Program (Valued at $12,000 over 4 years)

JOURNAL ARTICLES Vilk G, Weber JE, Turowec JP, Duncan JS, Wu C, Derksen DR, Zien P, Sarno S, Donella-Deana A, Lajoie G, Pinna LA, Litchfield DW (2008) Protein kinase CK2 catalyzes tyrosine phosphorylation in mammalian cells. Cell Signal, Jul 6. [Epub ahead of print], PMID: 18662771.

Duncan, JS, Gyenis, L, Lenehan, J, Graves, LM, Haystead, TA, Litchfield, DW (2008) An unbiased evaluation of CK2 inhibitors by chemo-proteomics: Characterization of inhibitor effects on CK2 and identification of novel inhibitor targets. Mol Cell Proteomics. Jun;7(6): 1077-88.

Duncan, JS and Litchfield, DW. (2008) Too much of a good thing: The role of CK2 in tumorigenesis and prospects for therapeutic intervention of CK2. Biochimica et Biophysica Acta - Proteins and Proteomics. Jan;1784(l):33-47.

Zien, P, Duncan, JS, Skierski, J, Bretner, M, Litchfield, DW, Shugar, D. (2005) Tetrabromobenzotriazole (TBBt) and tetrabromobenzimidizole (TBBz) as selective inhibitors of protein kinase CK2: Evaluation of their effects on cells and different molecular forms of human CK2. Biochimica et Biophysica Acta, Dec 30;1754(l-2): 271-280.

CONFERENCE PRESENTATIONS

Duncan JS, Turowec JP, and Litchfield DW. (2008) The Role of CK2 in Caspase Signaling. The Dynactome-2nd Annual Meeting, Toronto, Canada, February 20th.

-Invited oral speaker in the in vitro domain mapping symposia 195

Duncan JS, and Litchfield DW. (2007) Proteome-Mining: a Search for CK2 Substrates. 5th International Conference in Protein Kinases, Warsaw, Poland, June 23-28, 2007. - Invited oral speaker in the protein kinase CK2 symposia

Duncan JS, and Litchfield DW. (2007) Working Towards Knowledge Based Cancer Therapeutics. Daffodil Month Kick-off Breakfast, Canadian Cancer Society. Sarnia, Canada, March 22, 2007. -Invited keynote speaker in the "new drug targets " symposia

Duncan JS, and Litchfield DW. (2007) Working Towards Knowledge Based Cancer Therapeutics. Trillium Cancer Camp Teen Conference. Simcoe, Canada, March 17, 2007. -Invited oral speaker

Duncan JS, and Litchfield DW. (2006) Investigating the Efficacy of CK2 Inhibitors Using Functional Proteomics. LRCP & UWO Dept. of Oncology Research and Education Day, June 9, 2006. -Invited oral speaker

ABSTRACTS

Duncan JS, Gyenis L and Litchfield DW. (2007) Characterization of CK2 inhibitors using functional proteomics. Abstract presented at the 5th International Conference of Inhibitors of Protein Kinases. Warsaw, Poland, June 23-28.

Duncan JS and Litchfield DW. (2006) Investigating the efficacy of CK2-dependent inhibitors: Evidence for a role of CK2 in apoptosis. Abstract presented at the ASBMB Annual Meeting and Centennial Celebration. San Francisco, USA, April 1-4. 196

Zien P, Duncan JS, Litchfield DW, Skierski J, Bretner M, Shugar D. (2005). Inhibitors of protein kinase CK2 and their influence on activities of different forms of human CK2, and the cell cycle. Abstract presented by Piotr Zien at the 4th International Conference of Inhibitors of Protein Kinases and Workshop on Modeling of Specific Molecular Recognition Processes. Warsaw, Poland, June 25-29,2005. Duncan JS, and Litchfield DW. (2005) Investigating the role of CK2 in cell survival. Abstract presented at the LRCP & UWO, Dept. of Oncology Research and Education

Day, May 27, 2005.

Duncan JS, and Litchfield DW. (2005) Investigating the role of CK2 in cell survival. Abstract presented at the Margret P. Moffat Graduate Research Day, May 10, 2005.

UNIVERSITY SERVICES

2003-2004 Biochemistry Social Committee

EMPLOYMENT

2005-2007 Teaching Assistant (TA)- University of Western

Ontario (04-09) 2003 Research Associate- University of Western Ontario (04-09) 2000 Research Student- Dow Chemical Canada

REFERENCES

Dr. David W. Litchfield- supervisor for Ph.D. studies at the University of Western Ontario (Professor, Department of Biochemistry, University of Western Ontario) litchfi(g),uwo.ca 1-519-661-4186

Dr. Tim Haystead- collaborator and mentor (Associate professor, Department of Pharmacology, Duke University Medical Center, Durham, NC) haystOO 1 ©mc.duke.edu 197

1-919-613-8606/9

Dr. Lee M Graves- collaborator and mentor (Associate professor, Department of Pharmacology, University of North Carolina at Chapel Hill, NC) lmg(g),med.unc.edu 1-919-966-0915